Binding polypeptides for B lymphocyte stimulator protein (BLyS)

ABSTRACT

Binding polypeptides comprising specific amino acid sequences are disclosed that bind B Lymphocyte Stimulator (BLyS) protein or BLyS-like polypeptides. The binding polypeptides can be used in methods of the invention for detecting or isolating BLyS protein or BLyS-like polypeptides in solutions or mixtures, such as blood, tissue samples, or conditioned media.

FIELD OF THE INVENTION

[0001] The present invention relates to polypeptides that bind to B lymphocyte stimulator protein (BLyS). Such polypeptides have uses for example, in the detection, isolation, and/or purification of BLyS. The invention also relates to nucleic acid molecules encoding these BLyS binding polypeptides, vectors and host cells containing these nucleic acids, and methods for producing the same.

BACKGROUND OF THE INVENTION

[0002] B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (“TNF”) superfamily that induces both in vivo and in vitro B cell proliferation and differentiation (Moore et al., Science, 285: 260-263 (1999)). BLyS is distinguishable from other B cell growth and differentiation factors such as IL-2, IL-4, IL-5, IL-6, IL-7, IL-13, IL-15, CD40L, or CD27L (CD70) by its monocyte-specific gene and protein expression pattern and its specific receptor distribution and biological activity on B lymphocytes. BLyS expression is not detected on natural killer (“NK”) cells, T cells or B cells, but is restricted to cells of myeloid origin. BLyS expression on resting monocytes is upregulated by interferon-gamma (IFN-gamma). The gene encoding BLyS has been mapped to chromosome 13q34.

[0003] BLyS is expressed as a 285 amino acid type II membrane-bound polypeptide and a soluble 152 amino acid polypeptide (Moore et al., 1999, supra). The membrane-bound form of BLyS has a predicted transmembrane spanning domain between amino acid residues 47 and 73. The NH₂-terminus of the soluble form of BLyS begins at Ala¹³⁴ of the membrane-bound form of BLyS. Both the soluble and membrane-bound forms of the protein form homotrimers. Soluble recombinant BLyS has been shown to induce in vitro proliferation of murine splenic B cells and to bind to a cell-surface receptor on these cells (Moore et al., 1999, supra). Soluble BLyS administration to mice has been shown to result in an increase in the proportion of CD45R^(dull), Ly6D^(bright) (also known as ThB) B cells and an increase in serum IgM and IgA levels (Moore et al., 1999, supra). Thus, BLyS displays a B cell tropism in both its receptor distribution and biological activity.

[0004] Based on its expression pattern and biological activity, BLyS has been suggested to be involved in the exchange of signals between B cells and monocytes or their differentiated progeny. The restricted expression patterns of BLyS receptor and ligand suggest that BLyS may function as a regulator of T cell-independent responses in a manner analogous to that of CD40 and CD40L in T cell-dependent antigen activation.

[0005] Accordingly, molecules that specifically bind BLyS would find a variety of uses in the study of the BLyS cytokine, in the manufacture and purification of BLyS in commercial and medically pure quantities, and in the development new therapeutic or diagnostic reagents.

SUMMARY OF THE INVENTION

[0006] The present invention provides new polypeptides and families of polypeptides that specifically bind to B lymphocyte stimulator protein (BLyS) and/or BLyS-like polypeptides. In particular, the invention encompasses polypeptides that specifically bind to a polypeptide or polypeptide fragment of human BLyS (SEQ ID NOs: 173 and/or 174) or BLyS expressed on human monocytes; murine BLyS (SEQ ID NOs: 175 and/or 176) or BLyS expressed on murine monocytes; rat BLyS (either the soluble forms as given in SEQ ID NOs: 177, 178, 179 and/or 180 or in a membrane associated form, e.g., on the surface of rat monocytes); or monkey BLyS (e.g., the monkey BLyS polypeptides of SEQ ID NOS: 181 and/or 182, the soluble form of monkey BLyS, or BLyS expressed on monkey monocytes), preferably human BLyS.

[0007] In specific preferred embodiments, the BLyS binding polypeptides of the invention bind BLyS and/or BLyS-like polypeptides with high affinity. In other embodiments, the BLyS binding polypeptides of the invention reversibly bind BLyS and/or BLyS-like polypeptides. In still other embodiments, the BLyS binding polypeptides of the invention irreversibly bind BLyS and/or BLyS-like polypeptides.

[0008] The cysteine residues in certain polypeptides according to the invention are believed to form a disulfide bond, which would cause the polypeptide containing these cysteine residues to form a stable loop structure under non-reducing conditions. Especially preferred BLyS binding polypeptides of the invention are polypeptide molecules that comprise amino acid sequences that form stable loop structures or other stable structures that bind BLyS or BLyS-like polypeptides.

[0009] In specific embodiments, the invention relates to BLyS binding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12, 20-172, and 186-444, preferably SEQ ID NOs: 163-172 or 436-444 as referred to above and in Tables 1-8, 14 and 15 and in Examples 2, 5 and 6 below. Analysis of the sequences of the BLyS binding polypeptides isolated as described herein shows a strong selection for polypeptides containing the tetrapeptide Asp-Xaa-Leu-Thr (SEQ ID NO: 446), and therefore in its broadest aspects, the present invention relates to polypeptides capable of binding to BLyS comprising the polypeptide Asp-Xaa-Leu-Thr (SEQ ID NO: 446), where Xaa is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala (preferably Pro or Ser).

[0010] Seven consensus sequences (SEQ ID NOs: 1-7) have been determined based on the specific BLyS binding polypeptides shown in Tables 1-8. In specific embodiments, BLyS binding polypeptides of the invention comprise one or more of these sequences. Such preferred BLyS binding polypeptides include polypeptides with the potential to form a cyclic or loop structure between invariant Cys residues comprising, or alternatively consisting of, an amino acid sequence selected from A-E (SEQ ID NOs: 1-5):

[0011] (A) X₁-X₂-X₃-Cys-X₅-Phe-X₇-Trp-Glu-Cys-X₁₁-X₁₂-X₁₃ (SEQ ID NO: 1), wherein

[0012] X₁ is Ala, Asn, Lys, or Ser;

[0013] X₂ is Ala, Glu, Met, Ser, or Val;

[0014] X₃ is Ala, Asn, Lys, or Pro (preferably Lys);

[0015] X₅ is Phe, Trp, or Tyr (preferably Tyr);

[0016] X₇ is Pro or Tyr (preferably Pro);

[0017] X₁₁ is Ala, Gln, His, Phe, or Val;

[0018] X₁₂ is Asn, Gln, Gly, His, Ser, or Val; and

[0019] X₁₃ is Ala, Asn, Gly, Ile, Pro, or Ser, wherein said polypeptide binds BLyS and/or BLyS-like polypeptides; or

[0020] (B) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-Cys-X₁₂-X₁₃-x₁₄ (SEQ ID NO: 2), wherein

[0021] X₁ is Ala, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, or is absent;

[0022] X₂ is Ala, Asn, Asp, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0023] X₃ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val (preferably Asp);

[0024] X₅ is Asp, Ile, Leu, or Tyr (preferably Asp or Leu);

[0025] X₆ is Arg, Asp, Glu, His, Ile, Leu, Lys, Phe, Pro, Tyr, or Val (preferably Glu or Leu);

[0026] X₇ is His, Leu, Lys, or Phe (preferably His or Leu);

[0027] X₈ is Leu, Pro, or Thr (preferably Thr or Pro);

[0028] X₉ is Arg, Asn, Gly, His, Ile, Lys, Met, or Trp (preferably Lys);

[0029] X₁₀ is Ala, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Ser, Trp, Tyr, or Val;

[0030] X₁₂ is Asp, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Ser, Trp, Tyr, or Val;

[0031] X₁₃ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and

[0032] X₁₄ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Trp, Tyr, Val, or is absent, wherein said polypeptide binds BLyS and/or BLyS-like polypeptides; or

[0033] (C) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys-X₁₃-X₁₄-X₁₅ (SEQ ID NO: 3), wherein

[0034] X₁ is Ala, Arg, Asn, Asp, Leu, Lys, Phe, Pro, Ser, or Thr;

[0035] X₂ is Asn, Asp, Gln, His, Ile, Lys, Pro, Thr, or Trp;

[0036] X₃ is Ala, Arg, Asn, Gln, Glu, His, Phe, Pro, or Thr (preferably Ala);

[0037] X₅ is Asn, Asp, Pro, Ser, or Thr (preferably Asp);

[0038] X₆ is Arg, Asp, Ile, Leu, Met, Pro, or Val (preferably Ile);

[0039] X₇ is Ala, Ile, Leu, Pro, Thr, or Val (preferably Val or Leu);

[0040] X₈ is Asn, His, Ile, Leu, Lys, Phe, or Thr (preferably Thr);

[0041] X₉ is Asn, Glu, Gly, His, Leu, Lys, Met, Pro, or Thr (preferably Leu);

[0042] X₁₀ is Arg, Asn, Asp, Gln, Glu, Gly, Ile, Lys, Met, Pro, Ser, or Trp;

[0043] X₁₁ is Arg, Glu, Gly, Lys, Phe, Ser, Trp, or Tyr (preferably Ser);

[0044] X₁₃ is Gln, Glu, Ile, Leu, Phe, Pro, Ser, Tyr, or Val (preferably Val);

[0045] X₁₄ is Asn, Gly, Ile, Phe, Pro, Thr, Trp, or Tyr; and

[0046] X₁₅ is Asn, Asp, Glu, Leu, Lys, Met, Pro, or Thr (preferably Glu or Pro), wherein said polypeptide binds BLyS and/or BLyS-like polypeptides; or

[0047] (D) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-Cys-X₁₄-X₅-X₆ (SEQ ID NO: 4), wherein

[0048] X₁ is Asn, Asp, His, Leu, Phe, Pro, Ser, Tyr, or is absent (preferably Ser);

[0049] X₂ is Arg, Asn, Asp, His, Phe, Ser, or Trp (preferably Arg);

[0050] X₃ is Asn, Asp, Leu, Pro, Ser, or Val (preferably Asn or Asp);

[0051] X₅ is Asp, Gln, His, Ile, Leu, Lys, Met, Phe, or Thr;

[0052] X₆ is His, Ile, Leu, Met, Phe, Pro, Trp, or Tyr;

[0053] X₇ is Asp, His, Leu, or Ser (preferably Asp);

[0054] X₈ is Ala, Arg, Asp, Glu, Leu, Phe, Pro, or Thr (preferably Glu or Pro);

[0055] X₉ is Ala, Arg, Asn, or Leu (preferably Leu);

[0056] X₁₀ is Ile, Leu, Met, Pro, Ser, or Thr (preferably Thr);

[0057] X₁₁ is Ala, Arg, Asn, Gly, His, Lys, Ser, or Tyr;

[0058] X₁₂ is Ala, Arg, Asn, Gln, Leu, Met, Ser, Trp, Tyr, or Val;

[0059] X₁₄ is Asp, Gly, Leu, Phe, Tyr, or Val (preferably Leu);

[0060] X₁₅ is Asn, His, Leu, Pro, or Tyr (preferably His, Leu or Pro); and

[0061] X₁₆ is Asn, Asp, His, Phe, Ser, or Tyr, (preferably Asp or Ser), wherein said polypeptide binds BLyS and/or BLyS-like polypeptides; or

[0062] (E) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-Cys-X₁₆-X₁₇-X₁₈ (SEQ ID NO: 5), wherein

[0063] X₁ is Arg, Asp, Gly, His, Leu, Phe, Pro, Ser, Trp, Tyr, or is absent (preferably Arg);

[0064] X₂ is Ala, Arg, Asn, Asp, Gly, Pro, Ser, or is absent (preferably Asn, Asp, Gly, or Pro);

[0065] X₃ is Arg, Asn, Gln, Glu, Gly, Lys, Met, Pro, Trp or Val (preferably Gly or Met);

[0066] X₅ is Arg, Asn, Gln, Glu, His, Leu, Phe, Pro, Trp, Tyr, or Val (preferably Trp, Tyr, or Val);

[0067] X₆ is Arg, Asp, Gln, Gly, Ile, Lys, Phe, Thr, Trp or Tyr (preferably Asp);

[0068] X₇ is Ala, Arg, Asp, Glu, Gly, Leu, Ser, or Tyr (preferably Asp);

[0069] X₈ is Asp, Gln, Glu, Leu, Met, Phe, Pro, Ser, or Tyr (preferably Leu);

[0070] X₉ is Asp, Leu, Pro, Thr, or Val (preferably Leu or Thr);

[0071] X₁₀ is Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp or Tyr (preferably Lys or Thr);

[0072] X₁₁ is Ala, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, or Thr (preferably Arg or Leu);

[0073] X₁₂ is Ala, Asn, Gln, Gly, Leu, Lys, Phe, Pro, Thr, Trp, or Tyr (preferably Thr or Trp);

[0074] X₁₃ is Ala, Arg, Gln, His, Lys, Met, Phe, Pro, Thr, Trp, or Tyr (referably Met or Phe);

[0075] X₁₄ is Arg, Gln, Glu, Gly, His, Leu, Met, Phe, Pro, Ser, Thr, Tyr, or Val (preferably Val);

[0076] X₁₆ is Arg, Asp, Gly, His, Lys, Met, Phe, Pro, Ser, or Trp (preferably Met);

[0077] X₁₇ is Arg, Asn, Asp, Gly, His, Phe, Pro, Ser, Trp or Tyr, (preferably Arg, His, or Tyr); and

[0078] X₁₈ is Ala, Arg, Asn, Asp, His, Leu, Phe, or Trp (preferably His or Asn), wherein said polypeptide binds BLyS and/or BLyS-like polypeptides.

[0079] Additional preferred embodiments include linear polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from F and G (SEQ ID NOs: 6 and 7):

[0080] (F) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂ (SEQ ID NO: 6), wherein

[0081] X₁ is Ala, Arg, Gly, His, Leu, Lys, Met, Phe, Trp, Tyr, or Val (preferably Gly, Tyr, or Val);

[0082] X₂ is Ala, Arg, Gln, His, Ile, Leu, Phe, Thr, Trp, or Tyr (preferably His or Tyr);

[0083] X₃ is Ala, Asp, Lys, Phe, Thr, Trp or Tyr (preferably Asp or Tyr);

[0084] X₄ is Arg, Asp, Gln, Lys, Met, Phe, Pro, Ser, Tyr, or Val (preferably Asp or Gln);

[0085] X₅ is Asp, Leu, Lys, Phe, Pro, Ser, or Val (preferably Leu or Ser);

[0086] X₆ is His, Ile, Leu, Pro, Ser, or Thr (preferably Leu or Thr);

[0087] X₇ is Arg, Gly, His, Leu, Lys, Met, or Thr (preferably Lys or Thr);

[0088] X₈ is Ala, Arg, Asn, Ile, Leu, Lys, Met, or Thr (preferably Leu or Lys);

[0089] X₉ is Ala, Asn, Arg, Asp, Glu, Gly, His, Leu, Met, Ser, Trp, Tyr, or Val (preferably Met or Ser);

[0090] X₁₀ is Ile, Leu, Phe, Ser, Thr, Trp, Tyr, or Val (preferably Thr or Leu);

[0091] X₁₁ is Ala, Arg, Gly, His, Ile, Leu, Lys, Pro, Ser, Thr, Trp, Tyr, or Val (preferably Pro or Thr); and

[0092] X₁₂ is Arg, Asp, His, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val (preferably Arg or Pro), wherein said polypeptide binds BLyS and/or BLyS-like polypeptides; or

[0093] (G) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃ (SEQ ID NO: 7), wherein

[0094] X₁ is Asp, Gln, Glu, Gly, His, Lys, Met, or Trp (preferably Glu, Lys);

[0095] X₂ is Arg, Gln, His, Ile, Leu, or Pro (preferably His or Pro);

[0096] X₃ is Asp, Gly, Ile, Lys, Thr, Tyr or Val (preferably Tyr);

[0097] X₄ is Asn, Asp, Gln, Glu, Met, Pro, Ser, or Tyr (preferably Asp or Gln);

[0098] X₅ is Asn, Asp, His, Ile, Leu, Met, Pro, Thr or Val (preferably Asn or Thr);

[0099] X₆ is Asp, Glu, His, Leu, Lys, Pro, or Val (preferably Asp or Pro);

[0100] X₇ is Arg, Asn, Gln, His, Ile, Leu, Met, Pro, or Thr (preferably Ile or Pro);

[0101] X₈ is Gln, Gly, His, Leu, Met, Ser, or Thr (preferably Leu or Thr);

[0102] X₉ is Asn, Gln, Gly, His, Leu, Lys, Ser, or Thr (preferably Lys);

[0103] X₁₀ is Ala, Gly, Ile, Leu, Lys, Met, or Phe (preferably Gly or Met);

[0104] X₁₁ is Ala, Glu, His, Ile, Leu, Met, Ser, Thr, Trp, Tyr, or Val (preferably Ala or Thr);

[0105] X₁₂ is Arg, Gln, Glu, Gly, His, Ile, Lys, Tyr, or Val (preferably Arg or His); and

[0106] X₁₃ is Arg, Asn, Glu, His, Ile, Ser, Thr, Trp, or Val (preferably His), wherein said polypeptide binds BLyS and/or BLyS-like polypeptides.

[0107] Said polypeptides may have additional amino acids attached at either or both of the N- and C-terminal ends.

[0108] Examination of the sequence information and binding data from the isolates of libraries containing polypeptides with the potential to form loop structures (i.e., libraries designated TN6, TN7, TN8, TN9, TN10 and TN12) identifies a series of BLyS binding polypeptides that may form loop structures. In specific embodiments, BLyS binding polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from H-L (SEQ ID NOs: 8-12):

[0109] (H) Cys-X₂-Phe-X₄-Trp-Glu-Cys (SEQ ID NO: 8), wherein

[0110] X₂ is Phe, Trp, or Tyr (preferably Tyr); and

[0111] X₄ is Pro or Tyr (preferably Pro); or

[0112] (I) Cys-X₂-X₃-X₄-X₅-X₆-X₇-Cys (SEQ ID NO: 9), wherein

[0113] X₂ is Asp, Ile, Leu, or Tyr (preferably Asp or Leu);

[0114] X₃ is Arg, Asp, Glu, His, Ile, Leu, Lys, Phe, Pro, Tyr, or Val (preferably Glu or Leu);

[0115] X₄ is His, Leu, Lys, or Phe (preferably His or Leu);

[0116] X₅ is Leu, Pro, or Thr (preferably Thr or Pro);

[0117] X₆ is Arg, Asn, Gly, His, Ile, Lys, Met, or Trp (preferably Lys); and

[0118] X₇ is Ala, Asn, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Ser, Trp, Tyr, or Val; or

[0119] (J) Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-Cys (SEQ ID NO: 10), wherein

[0120] X₂ is Asn, Asp, Pro, Ser, or Thr (preferably Asp);

[0121] X₃ is Arg, Asp, Ile, Leu, Met, Pro, or Val (preferably Ile);

[0122] X₄ is Ala, Ile, Leu, Pro, Thr, or Val (preferably Val or Leu);

[0123] X₅ is Asn, His, Ile, Leu, Lys, Phe, or Thr (preferably Thr);

[0124] X₆ is Asn, Glu, Gly, His, Leu, Lys, Met, Pro, or Thr (preferably Leu);

[0125] X₇ is Arg, Asn, Asp, Gln, Glu, Gly, Ile, Lys, Met, Pro, Ser, or Trp;

[0126] X₈ is Arg, Glu, Gly, Lys, Phe, Ser, Trp, or Tyr (preferably Ser); or

[0127] (K) Cys-X₂-X₃-X₄-X₅-X₆-X₇X₈-X₉-Cys (SEQ ID NO: 11), wherein

[0128] X₂ is Asp, Gln, His, Ile, Leu, Lys, Met, Phe, or Thr;

[0129] X₃ is His, Ile, Leu, Met, Phe, Pro, Trp, or Tyr;

[0130] X₄ is Asp, His, Leu, or Ser (preferably Asp);

[0131] X₅ is Ala, Arg, Asp, Glu, Leu, Phe, Pro, or Thr (preferably Glu or Pro);

[0132] X₆ is Ala, Arg, Asn, or Leu (preferably Leu);

[0133] X₇ is Ile, Leu, Met, Pro, Ser, or Thr (preferably Thr);

[0134] X₈ is Ala, Arg, Asn, Gly, His, Lys, Ser, or Tyr;

[0135] X₉ is Ala, Arg, Asn, Gln, Leu, Met, Ser, Trp, Tyr, or Val; or

[0136] (L) Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys (SEQ ID NO: 12), wherein

[0137] X₂ is Arg, Asn, Gln, Glu, His, Leu, Phe, Pro, Trp, Tyr, or Val (preferably Trp, Tyr, or Val);

[0138] X₃ is Arg, Asp, Gln, Gly, Ile, Lys, Phe, Thr, Trp or Tyr (preferably Asp);

[0139] X₄ is Ala, Arg, Asp, Glu, Gly, Leu, Ser, or Tyr (preferably Asp);

[0140] X₅ is Asp, Gln, Glu, Leu, Met, Phe, Pro, Ser, or Tyr (preferably Leu);

[0141] X₆ is Asp, Leu, Pro, Thr, or Val (preferably Leu or Thr);

[0142] X₇ is Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp or Tyr (preferably Lys or Thr);

[0143] X₈ is Ala, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, or Thr (preferably Arg or Leu);

[0144] X₉ is Ala, Asn, Gln, Gly, Leu, Lys, Phe, Pro, Thr, Trp, or Tyr (preferably Thr or Trp);

[0145] X₁₀ is Ala, Arg, Gln, His, Lys, Met, Phe, Pro, Thr, Trp, or Tyr (preferably Met or Phe);

[0146] X₁₁ is Arg, Gln, Glu, Gly, His, Leu, Met, Phe, Pro, Ser, Thr, Tyr, or Val (preferably Val); wherein said polypeptides bind BLyS and/or BLyS-like polypeptides.

[0147] In additional preferred embodiments of the present invention, BLyS binding polypeptides comprise the following amino acid sequence M (SEQ ID NO: 447):

[0148] (M) Ala-X₂-X₃-X₄-Asp-X₆-Leu-Thr-X₉-Leu-X₁₁-X₁₂-X₁₃-X₁₄ (SEQ ID NO: 447), wherein

[0149] X₂ is Asn, Ser, Tyr, Asp, Phe, Ile, Gln, His, Pro, Lys, Leu, Met, Thr, Val, Glu, Ala, Gly, Cys, or Trp (i.e., any amino acid except Arg; preferably Asn);

[0150] X₃ is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly, or Ser (preferably Trp);

[0151] X₄ is Tyr, Phe, Glu, Cys, Asn (preferably Tyr);

[0152] X₆ is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala (preferably Pro or Ser);

[0153] X₉ is Lys, Asn, Gln, Gly, or Arg (preferably Lys);

[0154] X₁₁ is Trp, Ser, Thr, Arg, Cys, Tyr, or Lys (preferably Tip);

[0155] X₁₂ is Leu, Phe, Val, Ile, or His (preferably Leu);

[0156] X₁₃ is Pro, Leu, His, Ser, Arg, Asn, Gln, Thr, Val, Ala, Cys, Ile, Phe, or Tyr (i.e., not Asp, Glu, Gly, Lys, Met, or Trp; preferably Pro); and

[0157] X₁₄ is Asp, Glu, Asn, Val, His, Gln, Arg, Gly, Ser, Tyr, Ala, Cys, Lys, Ile, Thr or Leu (i.e., not Phe, Met, Pro, or Trp; preferably Asp, Val or Glu).

[0158] Preferred embodiments are polypeptides comprising a core sequence of the formula N:

[0159] (N) X₁-X₂-Asp-X₄-Leu-Thr-X₇-Leu-X₉-X₁₀ (SEQ ID NO: 448), wherein

[0160] X₁ is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly, or Ser (preferably Trp);

[0161] X₂ is Tyr, Phe, Glu, Cys, Asn (preferably Tyr);

[0162] X₄ is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala (preferably Pro or Ser);

[0163] X₇ is Lys, Asn, Gln, Gly, or Arg (preferably Lys);

[0164] X₉ is Trp, Ser, Thr, Arg, Cys, Tyr, or Lys (preferably Trp); and

[0165] X₁₀ is Leu, Phe, Val, Ile, or His (preferably Leu).

[0166] Especially preferred BLyS binding polypeptides according to the present invention comprise the core peptide Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu (SEQ ID NO: 436).

[0167] The BLyS binding polypeptides described above may have additional amino acids attached at either or both of the N- and C-terminal ends.

[0168] A further embodiment of the present invention relates to a BLyS affinity maturation library, comprising a population of at least 10³ polypeptides, preferably at least 10⁶ polypeptides, more preferably at least 10⁹ or more polypeptides, wherein the polypeptides of said population comprise the amino acid sequence:

[0169] Ala-X₂-X₃-X₄-Asp-X₆-Leu-Thr-X₉Leu-X₁₁-X₁₂-X₁₃-X₁₄ (SEQ ID NO: 449), wherein

[0170] X₂ is any amino acid;

[0171] X₃ is any amino acid;

[0172] X₄ is any amino acid;

[0173] X₆ is any amino acid;

[0174] X₉ is any amino acid;

[0175] X₁₁ is any amino acid;

[0176] X₁₂ is any amino acid;

[0177] X₁₃ is any amino acid; and

[0178] X₁₄ is any amino acid.

[0179] A preferred BLyS affinity maturation library will be produced such that the variable amino acid positions (i.e., positions 2, 3, 4, 6, 9, 11, 12, 13 and 14 in SEQ ID NO: 449) will not be randomly variegated but will disproportionately be a single selected amino acid. Such a library may be produced by expression of a multiplicity of polynucleotides fitting the DNA template:

[0180] GCT NNN NNN NNN GAT NNN CTT ACT NNN CTC NNN NNN NNN NNN (SEQ ID NO: 185), where each variable base (N) is A or C or G or T but one base in each instant is approximately 11-fold more probable at a given base position. One such DNA template, discussed in Example 6 below, is prepared so that the nucleotides of the DNA sequences are in the following proportions: in DNA template (SEQ ID NO:185) Proportion of Bases at Position Codon Base Position A C G T 2 4 79% 7% 7% 7% 5 79% 7% 7% 7% 6 7% 7% 7% 79% 3 7 7% 7% 7% 79% 8 7% 7% 79% 7% 9 7% 7% 79% 7% 4 10 7% 7% 7% 79% 11 79% 7% 7% 7% 12 7% 7% 7% 79% 6 16 7% 7% 7% 79% 17 7% 79% 7% 7% 18 7% 7% 7% 79% 9 25 79% 7% 7% 7% 26 79% 7% 7% 7% 27 7% 7% 79% 7% 11 31 7% 7% 7% 79% 32 7% 7% 79% 7% 33 7% 7% 79% 7% 12 34 7% 79% 7% 7% 35 7% 7% 7% 79% 36 7% 7% 7% 79% 13 37 7% 79% 7% 7% 38 7% 79% 7% 7% 39 7% 7% 7% 79% 14 40 7% 7% 79% 7% 41 79% 7% 7% 7% 42 7% 7% 7% 79%

[0181] BLyS binding polypeptide molecules of the invention may also have an amino terminal (N-terminal) capping or functional group, such as an acetyl group, which, for example, blocks the amino terminal amino group from undesirable reactions or is useful in linking the BLyS binding polypeptide to another molecule, matrix, resin, or solid support. BLyS binding polypeptides of the invention may also have a carboxy terminal (C-terminal) capping or functional group, such as an amide group, which, for example, blocks the C-terminal carboxyl group from undesirable reactions or provides a functional group useful in conjugating the binding polypeptide to other molecules, matrices, resins, or solid supports. Preferably, the N- and/or C-terminal capping groups are polypeptide linker molecules. An especially preferred C-terminal linker molecule that is useful for immobilizing a BLyS binding polypeptide of the invention to a solid support or chromatographic matrix material comprises the amino acid sequence Pro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ ID NO: 13). Another useful C-terminal linker, e.g., for fluoresceinating peptides, is Gly-Gly-Lys (see Table 15).

[0182] The invention also encompasses BLyS binding polypeptides that have been modified, for example, to increase or decrease the stability of the molecule, while retaining the ability to bind BLyS and/or BLyS-like polypeptides. An example of a modified BLyS binding polypeptide of the invention is a polypeptide in which one of two cysteine residues is substituted with a non-naturally occurring amino acid that is capable of condensing with the remaining cysteine side chain to form a stable thioether bridge, thereby generating a cyclic BLyS binding polypeptide. Such cyclic thioether molecules of synthetic peptides may be routinely generated using techniques known in the art, e.g., as described in PCT publication WO 97/46251, incorporated herein by reference.

[0183] In another embodiment, the invention provides BLyS binding polypeptides of the invention attached, coupled, linked or adhered to a matrix or resin or solid support. Techniques for attaching, linking or adhering polypeptides to matrices, resins and solid supports are well known in the art. Suitable matrices, resins or solid supports for these materials may be any composition known in the art to which a BLyS binding polypeptide of the invention could be attached, coupled, linked, or adhered, including but not limited to, a chromatographic resin or matrix, such as SEPHAROSE-4 FF agarose beads, the wall or floor of a well in a plastic microtiter dish, such as used in an enzyme-liked immunosorbent assay (ELISA), or a silica based biochip. Materials useful as solid supports on which to immobilize binding polypeptides of the invention include, but are not limited to, polyacrylamide, agarose, silica, nitrocellulose, paper, plastic, nylon, metal, and combinations thereof. A BLyS binding polypeptide of the invention may be immobilized on a matrix, resin or solid support material by a non-covalent association or by covalent bonding, using techniques known in the art. Preferably, a BLyS binding polypeptide of the invention is immobilized on a chromatography material such as SEPHAROSE-4 FF agarose. In an even more preferred embodiment, a BLyS binding polypeptide of the invention is coupled to a chromatography material using a linker molecule. A preferred linker molecule according to the present invention is a polypeptide comprising the amino acid sequence Pro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ ID NO: 13). Most preferably, the affinity chromatography material of the invention comprises a BLyS binding polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 163-172, which is linked to a chromatography material by a polypeptide linker molecule having the amino acid sequence Pro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ ID NO: 13). BLyS binding polypeptides, particularly attached, coupled, linked or adhered to a matrix or resin or other solid support are useful for methods of detecting, isolating and purifying BLyS and/or BLyS like polypeptides, particularly for purification of BLyS and/or BLyS like polypeptides by affinity chromatography.

[0184] In certain preferred embodiments, the BLyS binding polypeptides of the present invention or phage displaying such binding polypeptides, irreversibly bind the BLyS protein in its native, soluble trimeric form.

[0185] In certain preferred embodiments, the BLyS binding polypeptides of the present invention or phage displaying such binding polypeptides, reversibly bind the BLyS protein in its native, soluble trimeric form.

[0186] In a further embodiment, the present invention encompasses a composition of matter comprising isolated nucleic acids, preferably DNA, encoding a BLyS binding polypeptide of the invention. In a specific embodiment, nucleic acid molecules of the invention encode a BLyS binding polypeptide of the invention as provided in SEQ ID NOs: 1-12, 20-172, and 186-444. In additional embodiments, nucleic acid molecules of the invention encode a polypeptide variant or fragment of a polypeptide comprising an amino acid sequence of SEQ ID NOs: 1-12, 20-172, and 186-444. In a further additional embodiment, nucleic acid molecules of the invention encode a BLyS binding polypeptide, the complementary strand of which nucleic acid hybridizes to a polynucleotide sequence encoding a polypeptide described in Tables 1-8 and 14 and in Examples 2, 5 and 6 (SEQ ID NOs: 1-12, 20-172, and 186-444), under stringent conditions, e.g., hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate (SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDS at about 50-65° C., under highly stringent conditions, e.g., hybridization to filter-bound nucleic acid in 6×SSC at about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C., or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F. M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3).

[0187] The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules encoding the BLyS binding polypeptides of the present invention (as well as fragments and variants thereof), and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells. The invention further provides for the use of such recombinant vectors in the production of BLyS binding polypeptides by recombinant techniques.

[0188] The BLyS binding polypeptides, nucleic acids, transformed host cells, and genetically engineered viruses and phage of the invention (e.g., recombinant phage), have uses that include, but are not limited to, the detection, isolation, and purification of BLyS.

[0189] In another embodiment of the invention, recombinant bacteriophage displaying BLyS binding polypeptides on their surfaces are also provided. Such phage may be routinely generated using techniques known in the art and are useful, for example, as screening reagents and reagents for detecting BLyS.

[0190] In another embodiment, a BLyS binding polypeptide of the invention is used to detect or isolate BLyS or BLyS-like polypeptides in a solution. Such solutions include, but are not limited to, BLyS or BLyS-like polypeptides suspended or dissolved in water or a buffer solution as well as any fluid and/or cell obtained from an individual, biological fluid, body tissue, body cell, cell line, tissue culture, or other source which may contain BLyS or BLyS-like polypeptides, such as, cell culture medium, cell extracts, and tissue homogenates. Biological fluids include, but are not limited to, sera, plasma, lymph, blood, blood fractions, urine, synovial fluid, spinal fluid, saliva, and mucous.

[0191] In another embodiment, the present invention provides a method for detecting BLyS protein and/or BLyS-like polypeptide in a solution comprising, contacting the solution with a BLyS binding polypeptide of the invention and detecting binding of BLyS or BLyS-like polypeptide to the BLyS binding polypeptide. The BLyS binding polypeptide may be either free or immobilized. Preferably, the BLyS binding polypeptide is a polypeptide immobilized on a solid surface or chromatographic material or the well of a plastic microtiter assay dish.

[0192] Another embodiment of the present invention is a method for isolating BLyS protein and/or BLyS-like polypeptide from a solution containing it, comprising:

[0193] (a) contacting the solution with a BLyS binding polypeptide under conditions that permit binding of the BLyS and/or BLyS-like polypeptides to BLyS binding polypeptides, and

[0194] (b) separating BLyS binding polypeptides (and BLyS and/or BLyS-like polypeptides bound thereto) from the rest of the solution.

[0195] A further embodiment of the present invention is a method for isolating BLyS protein and/or BLyS-like polypeptide from a solution containing it, comprising:

[0196] (a) contacting the solution with a BLyS binding polypeptide under conditions that permit binding of the BLyS and/or BLyS-like polypeptides to BLyS binding polypeptides,

[0197] (b) separating the complex(es) formed by the BLyS binding polypeptide and BLyS and/or BLyS-like polypeptides from other components of the solution,

[0198] (c) dissociating the BLyS binding polypeptide from the BLyS and/or BLyS-like polypeptides, and

[0199] (d) recovering the dissociated, BLyS and/or BLyS-like polypeptides.

[0200] In another embodiment, the invention provides kits containing a binding polypeptide of the invention for use in methods of detecting or isolating BLyS and/or BLyS-like polypeptides.

[0201] Definitions

[0202] In order that the invention may be clearly understood, the following terms are defined:

[0203] The term “recombinant” is used to describe non-naturally altered or manipulated nucleic acids, host cells transfected with exogenous nucleic acids, or polypeptide molecules that are expressed non-naturally, through manipulation of isolated nucleic acid (typically, DNA) and transformation or transfection of host cells. “Recombinant” is a term that specifically encompasses nucleic acid molecules that have been constructed in vitro using genetic engineering techniques, and use of the term “recombinant” as an adjective to describe a molecule, construct, vector, cell, polypeptide or polynucleotide specifically excludes naturally occurring such molecules, constructs, vectors, cells, polypeptides or polynucleotides.

[0204] The term “bacteriophage” is defined as a bacterial virus containing a nucleic acid core and a protective shell built up by the aggregation of a number of different protein molecules. The terms “bacteriophage” and “phage” are synonymous and are used herein interchangeably.

[0205] The term “affinity ligand” is sometimes used herein and is synonymous with BLyS binding polypeptides of the invention.

[0206] The term “BLyS protein” as used herein encompasses both the membrane (e.g., SEQ ID NO: 173) and soluble forms (e.g., SEQ ID NO: 174). BLyS protein may be monomeric, dimeric, or trimeric or multivalent. Preferably, BLyS proteins are homotrimeric.

[0207] The term “BLyS-like polypeptide” as used herein encompasses natural BLyS or full-length recombinant BLyS as well as fragments and variants thereof, such as, a modified or truncated form of natural BLyS or fall-length recombinant BLyS, which BLyS and BLyS-like polypeptide retain a BLyS functional activity. BLyS or BLyS fragments that may be specifically bound by the compositions of the invention include, but are not limited to, human BLyS (SEQ ID NOs: 173 and/or 174) or BLyS expressed on human monocytes; murine BLyS (SEQ ID NOs: 175 and/or 176) or BLyS expressed on murine monocytes; rat BLyS (either the soluble forms as given in SEQ ID NOs: 177, 178, 179 and/or 180 or in a membrane associated form, e.g., on the surface of rat monocytes); or monkey BLyS (e.g., the monkey BLyS polypeptides of SEQ ID NOS: 181 and/or 182, the soluble form of monkey BLyS, or BLyS expressed on monkey monocytes) or fragments thereof. Preferably compositions of the invention bind human BLyS (SEQ ID NOs: 173 and/or 174) or fragments thereof. BLyS and BLyS-like polypeptides retain at least one functional activity of the natural or full-length BLyS, including but not limited to the following activities: binding to BLyS receptor (e.g., TACI (GenBank accesion number AAC51790), and BCMA (GenBank accession number NP_(—)001183)), stimulating B cell proliferation, stimulating immunoglobulin secretion by B cells, stimulating the BLyS receptor signaling cascade and/or being bound by an anti-BLyS antibody or other BLyS binding polypeptide. Assays that can be used to determine the functional activities of BLyS or BLyS like polypeptides can readily be determined by one skilled in the art (e.g., see assays disclosed in Moore et al., 1999, supra) “BLyS-like polypeptides” also include fusion polypeptides in which all or a portion of BLyS is fused or conjugated to another polypeptide. BLyS-like polypeptides that are fusion polypeptides retain at least one functional activity of BLyS, preferably the ability to stimulate B lymphocytes (see, for example, Moore et al., Science, 285: 260-263 (1999)), to bind the BLyS receptors (e.g., TACI or BCMA), and/or to be bound by an anti-BLyS antibody or other BLyS binding polypeptide. BLyS fusion polypeptides may be made by recombinant DNA techniques in which a gene or other polynucleotide coding sequence for BLyS or a fragment thereof is ligated in-frame (recombined) with the coding sequence of another protein or polypeptide. The resulting recombinant DNA molecule is then inserted into any of a variety of plasmid or phage expression vectors, which enable expression of the fusion protein molecule in an appropriate eukaryotic or prokaryotic host cell. BLyS fusion polypeptides may be generated by synthetic or semi-synthetic procedures as well.

[0208] The terms “BLyS target” or “BLyS target protein” are sometimes used herein and encompass BLyS and/or BLyS-like polypeptides. Thus, the BLyS binding polypeptides of the invention bind “BLyS target proteins” and can be used to bind, detect, remove, and/or purify “BLyS target proteins.”

[0209] The term “binding polypeptide” is used herein to refer to any polypeptide capable of forming a binding complex with another molecule, polypeptide, peptidomimetic or transformant.

[0210] A “BLyS binding polypeptide” is a molecule of the invention that can bind BLyS target protein. Non-limiting examples of BLyS binding polypeptides of the invention are the polypeptide molecules having an amino acid sequence described herein (see SEQ ID NOs: 1-12, 20-172, and 186-444). The term BLyS binding polypeptide also encompasses BLyS binding fragments and variants (including derivatives) of polypeptides having the specific amino acid sequences described herein (SEQ ID NOs: 1-12, 20-172, and 186-444). By “variant” of an amino acid sequence as described herein is meant a polypeptide that binds BLyS, but does not necessarily comprise an identical or similar amino acid sequence of a BLyS binding polypeptide specified herein. BLyS binding polypeptides of the invention which are variants of a BLyS binding polypeptide specified herein satisfy at least one of the following: (a) a polypeptide comprising, or alternatively consisting of, an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% least 99%, or 100% identical to the amino acid sequence of a BLyS binding polypeptide sequence disclosed herein (SEQ ID NOs: 1-12, 20-172, and 186-444), (b) a polypeptide encoded by a nucleotide sequence, the complementary sequence of which hybridizes under stringent conditions to a nucleotide sequence encoding a BLyS binding polypeptide disclosed herein (e.g., a nucleic acid sequence encoding the amino acid sequence of SEQ ID NOs: 1-12, 20-172, and 186-444), and/or a fragment of a BLyS binding polypeptide disclosed herein, of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, or at least 20 amino acid residues. BLyS binding polypeptides of the invention also encompass polypeptide sequences that have been modified for various applications provided that such modifications do not eliminate the ability to bind a BLyS target. Specific, non-limiting examples of modifications contemplated include C-terminal or N-terminal amino acid substitutions or peptide chain elongations for the purpose of linking the BLyS binder to a chromatographic material or other solid support. Other substitutions contemplated herein include substitution of one or both of a pair of cysteine residues that normally form disulfide links, for example with non-naturally occurring amino acid residues having reactive side chains, for the purpose of forming a more stable bond between those amino acid positions than the former disulfide bond. All such modified binding polypeptides are also considered BLyS binding polypeptides according to this invention so long as the modified polypeptides retain the ability to bind BLyS and/or BLyS-like polypeptides, and therefore, may be used in one or more of the various methods described herein, such as, to detect, purify, or isolate BLyS or BLyS-like polypeptides in or from a solution. BLyS binding polypeptides of the invention also include variants of the specific BLyS binding polypeptide sequences disclosed herein (e.g., SEQ ID NOs: 1-12, 20-172, and 186-444) which have an amino acid sequence corresponding to one of these polypeptide sequences, but in which the polypeptide sequence is altered by substitutions, additions or deletions that provide for molecules that bind BLyS. Thus, the BLyS binding polypeptides include polypeptides containing, as a primary amino acid sequence, all or part of the particular BLyS binding polypeptide sequence including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence, resulting in a peptide which is functionally active. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Conservative substitutions for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such BLyS binding polypeptides can be made either by chemical peptide synthesis or by recombinant production from a nucleic acid encoding the BLyS binding polypeptide which nucleic acid has been mutated. Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson et al., J. Biol. Chem., 253:6551 (1978)), use of TAB.RTM. linkers (Pharmacia), etc.

[0211] As used and understood herein, percent homology or percent identity of two amino acid lo sequences or of two nucleic acid sequences is determined using the algorithm of Karlin and Atschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268 (1990)), modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993)). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol., 215: 403-410 (1990)). BLAST nucleotide searches are performed with the NBLAST program to obtain nucleotide sequences homologous to a nucleic acid molecule described herein. BLAST protein searches are performed with the XBLAST program to obtain amino acid sequences homologous to a reference polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (Nucleic Acids Res., 25: 3389-3402 (1997)). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See, http://www.ncbi.nlm.nih.gov. Alternatively, the percent identity of two amino acid sequences or of two nucleic acid sequences can be determined once the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions/total number of positions×100%). In one embodiment, the two sequences are the same length.

[0212] The term “polypeptide”, as used herein, refers to a linear, branched, or cyclic (e.g., containing a loop structure) polymer of two or more amino acid residues linked with a peptide bond. The term “polypeptide” is not restricted to any particular upper limit of amino acid residues. Thus, the BLyS affinity ligands of the invention that comprise an amino acid sequence described herein are properly referred to as “BLyS binding polypeptides” because such binding polypeptides contain at least two amino acid residues held together by a peptide bond, even though such molecules may also contain one or more additional moieties or groups that are not amino acids, such as N-terminal and/or C-terminal capping or functional groups, and that may or may not be involved in a peptide bond. The polypeptides of the invention may be monovalent, divalent, trivalent, or multivalent and may comprise one or more of the BLyS binding polypeptides having the amino acid sequence of SEQ ID NOs: 1-12, 20-172, and 186-444 and/or fragments or variants thereof. The term “peptide” is used herein to have the same meaning as “polypeptide.”

[0213] “Feed stream”: BLyS and BLyS-like polypeptides that are bound by a BLyS binding polypeptide of this invention may be produced by any method known in the art, including, but not limited to, chemical synthesis; production in transformed host cells; secretion into culture medium by naturally occurring cells or recombinantly transformed bacteria, yeasts, fungi, insect cells, plant cells, and mammalian cells; production in genetically engineered organisms (for example, transgenic mammals); and production in non-genetically engineered organisms. The solution, sample, or mixture that contains a BLyS or BLyS-like polypeptide as it is produced or is found present in a production solution will sometimes be referred to as the “feed stream”.

[0214] The term “binding” refers to the determination by standard techniques that a binding polypeptide recognizes and binds to a given target. Such standard techniques include, but are not limited to, affinity chromatography, equilibrium dialysis, gel filtration, enzyme linked immunosorbent assay (ELISA), FACS analysis, and the monitoring of spectroscopic changes that result from binding, e.g., using fluorescence anisotropy, either by direct binding measurements or competition assays with another binder.

[0215] The term “specificity” refers to a binding polypeptide of the invention that has a higher binding affinity for one target over another. Thus, the term “BLyS target protein specificity” refers to a molecule having a higher affinity for BLyS target protein as compared with another molecule that is not a BLyS target protein.

[0216] Other terms are defined as necessary in the text below.

DETAILED DESCRIPTION OF THE INVENTION

[0217] The present invention provides novel binding moieties for BLyS. Such binding moieties make possible the efficient detection and isolation of BLyS or BLyS-like polypeptides in tissues or in a solution or system that contains BLyS or BLyS-like polypeptides. The BLyS binding polypeptides disclosed herein can also be used to immobilize BLyS targets and provide a means of removing BLyS target proteins from solutions or systems containing them. The preferred binding moieties of the present invention bind BLyS with high affinity, i.e., acting at low concentrations.

[0218] BLyS Binding Polypeptides

[0219] The present invention provides new polypeptides and families of polypeptides that specifically bind to B lymphocyte stimulator protein (BLyS) and/or BLyS-like polypeptides. In particular, the invention encompasses polypeptides that specifically bind to a polypeptide or polypeptide fragment of human BLyS (SEQ ID NOs: 173 and/or 174) or BLyS expressed on human monocytes; murine BLyS (SEQ ID NOs: 175 and/or 176) or BLyS expressed on murine monocytes; rat BLyS (either the soluble forms as given in SEQ ID NOs: 177, 178, 179 and/or 180 or in a membrane associated form, e.g., on the surface of rat monocytes); or monkey BLyS (e.g., the monkey BLyS polypeptides of SEQ ID NOS: 181 and/or 182, the soluble form of monkey BLyS, or BLyS expressed on monkey monocytes); preferably human BLyS.

[0220] In specific preferred embodiments, the BLyS binding polypeptides of the invention bind BLyS and/or BLyS-like polypeptides with high affinity. In other embodiments, the BLyS binding polypeptides of the invention reversibly bind BLyS and/or BLyS-like polypeptides. In still other embodiments, the BLyS binding polypeptides of the invention irreversibly bind BLyS and/or BLyS-like polypeptides.

[0221] The cysteine residues in polypeptides are believed to form a disulfide bond, which would cause the polypeptide containing these cysteine residues to form a stable loop structure under non-reducing conditions. Especially preferred BLyS binding polypeptides of the invention are polypeptide molecules that comprise amino acid sequences that form stable loop structures or other stable structures that bind BLyS or BLyS-like polypeptides.

[0222] In specific embodiments, the invention relates to BLyS binding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12, 20-172, and 186-444, preferably SEQ ID NOs: 163-172 or 436-444 as referred to above and in Tables 1-8, 14 and 15 and in Examples 2, 5 and 6 below. Analysis of the sequences of the BLyS binding polypeptides isolated as described herein shows a strong selection for polypeptides containing the tetrapeptide Asp-Xaa-Leu-Thr (SEQ ID NO: 446), and therefore in its broadest aspects, the present invention relates to polypeptides capable of binding to BLyS comprising the polypeptide Asp-Xaa-Leu-Thr (SEQ ID NO: 446), where Xaa is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala (preferably Pro or Ser).

[0223] Seven consensus sequences (SEQ ID NOs: 1-7) have been determined based on the specific BLyS binding polypeptides shown in Tables 1-8. In specific embodiments, BLyS binding polypeptides of the invention comprise one or more of these sequences. Such preferred BLyS binding polypeptides include polypeptides with the potential to formi a cyclic or loop structure between invariant Cys residues comprising, or alternatively consisting of, an amino Ad acid sequence selected from A-E (SEQ ID NOs: 1-5):

[0224] (A) X₁-X₂-X₃-Cys-X₅-Phe-X₇-Trp-Glu-Cys-X₁₁-X₁₂-X₁₃ (SEQ ID NO: 1), wherein

[0225] X₁ is Ala, Asn, Lys, or Ser;

[0226] X₂ is Ala, Glu, Met, Ser, or Val;

[0227] X₃ is Ala, Asn, Lys, or Pro preferably Lys);

[0228] X₅ is Phe, Trp, or Tyr (preferably Tyr);

[0229] X₇ is Pro or Tyr (preferably Pro);

[0230] X₁₁ is Ala, Gln, His, Phe, or Val;

[0231] X₁₂ is Asn, Gln, Gly, His, Ser, or Val; and

[0232] X₁₃ is Ala, Asn, Gly, Ile, Pro, or Ser, wherein said polypeptide binds BLyS and/or BLyS-like polypeptides; or

[0233] (B) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-Cys-X₁₂-X₁₃-X₁₄ (SEQ ID NO: 2), wherein

[0234] X₁ is Ala, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, or is absent;

[0235] X₂ is Ala, Asn, Asp, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0236] X₃ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val (preferably Asp);

[0237] X₅ is Asp, Ile, Leu, or Tyr (preferably Asp or Leu);

[0238] X₆ is Arg, Asp, Glu, His, Ile, Leu, Lys, Phe, Pro, Tyr, or Val (preferably Glu or Leu);

[0239] X₇ is His, Leu, Lys, or Phe (preferably His or Leu);

[0240] X₈ is Leu, Pro, or Thr (preferably Thr or Pro);

[0241] X₉ is Arg, Asn, Gly, His, Ile, Lys, Met, or Trp (preferably Lys);

[0242] X₁₀ is Ala, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Ser, Trp, Tyr, or Val;

[0243] X₁₂ is Asp, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Ser, Trp, Tyr, or Val;

[0244] X₁₃ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and

[0245] X₁₄ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Trp, Tyr, Val, or is absent, wherein said polypeptide binds BLyS and/or BLyS-like polypeptides; or .

[0246] (C) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys-X₁₃-X₁₄-X₁₅ (SEQ ID NO: 3); wherein

[0247] X₁ is Ala, Arg, Asn, Asp, Leu, Lys, Phe, Pro, Ser, or Thr;

[0248] X₂ is Asn, Asp, Gln, His, Ile, Lys, Pro, Thr, or Trp;

[0249] X₃ is Ala, Arg, Asn, Gln, Glu, His, Phe, Pro, or Thr (preferably Ala);

[0250] X₅ is Asn, Asp, Pro, Ser, or Thr (preferably Asp);

[0251] X₆ is Arg, Asp, Ile, Leu, Met, Pro, or Val (preferably Ile);

[0252] X₇ is Ala, Ile, Leu, Pro, Thr, or Val (preferably Val or Leu);

[0253] X₈ is Asn, His, Ile, Leu, Lys, Phe, or Thr (preferably Thr);

[0254] X₉ is Asn, Glu, Gly, His, Leu, Lys, Met, Pro, or Thr (preferably Leu);

[0255] X₁₀ is Arg, Asn, Asp, Gln, Glu, Gly, Ile, Lys, Met, Pro, Ser, or Trp;

[0256] X₁₁ is Arg, Glu, Gly, Lys, Phe, Ser, Trp, or Tyr (preferably Ser);

[0257] X₁₃ is Gln, Glu, Ile, Leu, Phe, Pro, Ser, Tyr, or Val (preferably Val);

[0258] X₁₄ is Asn, Gly, Ile, Phe, Pro, Thr, Trp, or Tyr; and

[0259] X₁₅ is Asn, Asp, Glu, Leu, Lys, Met, Pro, or Thr (preferably Glu or Pro), wherein said polypeptide binds BLyS and/or BLyS-like polypeptides; or

[0260] (D) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-Cys-X₁₄-X₁₅-X₁₆ (SEQ ID NO: 4), wherein

[0261] X₁ is Asn, Asp, His, Leu, Phe, Pro, Ser, Tyr, or is absent (preferably Ser);

[0262] X₂ is Arg, Asn, Asp, His, Phe, Ser, or Trp (preferably Arg);

[0263] X₃ is Asn, Asp, Leu, Pro, Ser, or Val (preferably Asn or Asp);

[0264] X₅ is Asp, Gln, His, Ile, Leu, Lys, Met, Phe, or Thr;

[0265] X₆ is His, Ile, Leu, Met, Phe, Pro, Trp, or Tyr;

[0266] X₇ is Asp, His, Leu, or Ser (preferably Asp);

[0267] X₈ is Ala, Arg, Asp, Glu, Leu, Phe, Pro, or Thr preferably Glu or Pro);

[0268] X₉ is Ala, Arg, Asn, or Leu (preferably Leu);

[0269] X₁₀ is Ile, Leu, Met, Pro, Ser, or Thr (preferably Thr);

[0270] X₁₁ is Ala, Arg, Asn, Gly, His, Lys, Ser, or Tyr;

[0271] X₁₂ is Ala, Arg, Asn, Gln, Leu, Met, Ser, Trp, Tyr, or Val;

[0272] X₁₄ is Asp, Gly, Leu, Phe, Tyr, or Val (preferably Leu);

[0273] X₁₅ is Asn, His, Leu, Pro, or Tyr (preferably His, Leu or Pro); and

[0274] X₁₆ is Asn, Asp, His, Phe, Ser, or Tyr, (preferably Asp or Ser), wherein said polypeptide binds BLyS and/or BLyS-like polypeptides; or

[0275] (E) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-Cys-X₁₆-X₁₇-X₁₈ (SEQ ID NO: 5), wherein

[0276] X₁ is Arg, Asp, Gly, His, Leu, Phe, Pro, Ser, Tip, Tyr, or is absent (preferably Arg);

[0277] X₂ is Ala, Arg, Asn, Asp, Gly, Pro, Ser, or is absent (preferably Asn, Asp, Gly, or Pro);

[0278] X₃ is Arg, Asn, Gln, Glu, Gly, Lys, Met, Pro, Trp or Val (preferably Gly or Met);

[0279] X₅ is Arg, Asn, Gln, Glu, His, Leu, Phe, Pro, Trp, Tyr, or Val (preferably Trp, Tyr, or Val);

[0280] X₆ is Arg, Asp, Gln, Gly, Ile, Lys, Phe, Thr, Trp or Tyr (preferably Asp);

[0281] X₇ is Ala, Arg, Asp, Glu, Gly, Leu, Ser, or Tyr (preferably Asp);

[0282] X₈ is Asp, Gln, Glu, Leu, Met, Phe, Pro, Ser, or Tyr (preferably Leu);

[0283] X₉ is Asp, Leu, Pro, Thr, or Val (preferably Leu or Thr);

[0284] X₁₀ is Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp or Tyr (preferably Lys or Thr);

[0285] X₁₁ is Ala, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, or Thr (preferably Arg or Leu);

[0286] X₁₂ is Ala, Asn, Gln, Gly, Leu, Lys, Phe, Pro, Thr, Trp, or Tyr (preferably Thr or Trp);

[0287] X₁₃ is Ala, Arg, Gln, His, Lys, Met, Phe, Pro, Thr, Trp, or Tyr (preferably Met or Phe);

[0288] X₁₄ is Arg, Gln, Glu, Gly, His, Leu, Met, Phe, Pro, Ser, Thr, Tyr, or Val (preferably Val);

[0289] X₁₆ is Arg, Asp, Gly, His, Lys, Met, Phe, Pro, Ser, or Trp (preferably Met);

[0290] X₁₇ is Arg, Asn, Asp, Gly, His, Phe, Pro, Ser, Trp or Tyr, (preferably Arg, His, or Tyr); and

[0291] X₁₈ is Ala, Arg, Asn, Asp, His, Leu, Phe, or Trp (preferably His or Asn), wherein said polypeptide binds BLyS and/or BLyS-like polypeptides.

[0292] Additional preferred embodiments include linear polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from F and G (SEQ ID NOs: 6 and 7):

[0293] (F) X₁-X₂₋X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂ (SEQ ID NO: 6), wherein

[0294] X₁ is Ala, Arg, Gly, His, Leu, Lys, Met, Phe, Trp, Tyr, or Val (preferably Gly, Tyr, or Val);

[0295] X₂ is Ala, Arg, Gln, His, Ile, Leu, Phe, Thr, Trp, or Tyr (preferably His or Tyr);

[0296] X₃ is Ala, Asp, Lys, Phe, Thr, Trp or Tyr (preferably Asp or Tyr);

[0297] X₄ is Arg, Asp, Gln, Lys, Met, Phe, Pro, Ser, Tyr, or Val (preferably Asp or Gln);

[0298] X₅ is Asp, Leu, Lys, Phe, Pro, Ser, or Val (preferably Leu or Ser);

[0299] X₆ is His, Ile, Leu, Pro, Ser, or Thr (preferably Leu or Thr);

[0300] X₇ is Arg, Gly, His, Leu, Lys, Met, or Thr (preferably Lys or Thr);

[0301] X₈ is Ala, Arg, Asn, Ile, Leu, Lys, Met, or Thr (preferably Leu or Lys);

[0302] X₉ is Ala, Asn, Arg, Asp, Glu, Gly, His, Leu, Met, Ser, Trp, Tyr, or Val (preferably Met or Ser);

[0303] X₁₀ is Ile, Leu, Phe, Ser, Thr, Trp, Tyr, or Val (preferably Thr or Leu);

[0304] X₁₁ is Ala, Arg, Gly, His, Ile, Leu, Lys, Pro, Ser, Thr, Trp, Tyr, or Val (preferably Pro or Thr); and

[0305] X₁₂ is Arg, Asp, His, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val (preferably Arg or Pro), wherein said polypeptide binds BLyS and/or BLyS-like polypeptides; or

[0306] (G) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃ (SEQ ID NO: 7), wherein

[0307] X₁ is Asp, Gln, Glu, Gly, His, Lys, Met, or Trp (preferably Glu, Lys);

[0308] X₂ is Arg, Gln, His, Ile, Leu, or Pro (preferably His or Pro);

[0309] X₃ is Asp, Gly, Ile, Lys, Thr, Tyr or Val (preferably Tyr);

[0310] X₄ is Asn, Asp, Gln, Glu, Met, Pro, Ser, or Tyr (preferably Asp or Gln);

[0311] X₅ is Asn, Asp, His, Ile, Leu, Met, Pro, Thr or Val (preferably Asn or Thr);

[0312] X₆ is Asp, Glu, His, Leu, Lys, Pro, or Val (preferably Asp or Pro);

[0313] X₇ is Arg, Asn, Gln, His, Ile, Leu, Met, Pro, or Thr (preferably Ile or Pro);

[0314] X₈ is Gln, Gly, His, Leu, Met, Ser, or Thr (preferably Leu or Thr);

[0315] X₉ is Asn, Gln, Gly, His, Leu, Lys, Ser, or Thr (preferably Lys);

[0316] X₁₀ is Ala, Gly, Ile, Leu, Lys, Met, or Phe (preferably Gly or Met);

[0317] X₁₁ is Ala, Glu, His, Ile, Leu, Met, Ser, Thr, Trp, Tyr, or Val (preferably Ala or Thr);

[0318] X₁₂ is Arg, Gln, Glu, Gly, His, Ile, Lys, Tyr, or Val (preferably Arg or His); and

[0319] X₁₃ is Arg, Asn, Glu, His, Ile, Ser, Thr, Trp, or Val (preferably His), wherein said polypeptide binds BLyS and/or BLyS-like polypeptides.

[0320] Said polypeptides may have additional amino acids attached at either or both of the N- and C-terminal ends.

[0321] Examination of the sequence information and binding data from the isolates of libraries containing polypeptides with the potential to form loop structures (i.e., libraries designated TN6, TN7, TN8, TN9, TN10 and TN12) identifies a series of BLyS binding polypeptides that may form loop structures. In specific embodiments, BLyS binding polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from H-L (SEQ ID NOs: 8-12):

[0322] (H) Cys-X₂-Phe-X₄Trp-Glu-Cys (SEQ ID NO: 8), wherein

[0323] X₂ is Phe, Trp, or Tyr (preferably Tyr); and

[0324] X₄ is Pro or Tyr (preferably Pro); or

[0325] (I) Cys-X₂-X₃-X₄-X₅-X₆-X₇-Cys (SEQ ID NO: 9), wherein

[0326] X₂ is Asp, Ile, Leu, or Tyr (preferably Asp or Leu);

[0327] X₃ is Arg, Asp, Glu, His, Ile, Leu, Lys, Phe, Pro, Tyr, or Val (preferably Glu or Leu);

[0328] X₄ is His, Leu, Lys, or Phe (preferably His or Leu);

[0329] X₅ is Leu, Pro, or Thr (preferably Thr or Pro);

[0330] X₆ is Arg, Asn, Gly, His, Ile, Lys, Met, or Trp (preferably Lys); and

[0331] X₇ is Ala, Asn, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Ser, Trp, Tyr, or Val; or

[0332] (J) Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-Cys (SEQ ID NO: 10), wherein

[0333] X₂ is Asn, Asp, Pro, Ser, or Thr (preferably Asp);

[0334] X₃ is Arg, Asp, Ile, Leu, Met, Pro, or Val (preferably Ile);

[0335] X₄ is Ala, Ile, Leu , Pro, Thr, or Val (preferably Val or Leu);

[0336] X₅ is Asn, His, Ile, Leu, Lys, Phe, or Thr (preferably Thr);

[0337] X₆ is Asn, Glu, Gly, His, Leu, Lys, Met, Pro, or Thr (preferably Leu);

[0338] X₇ is Arg, Asn, Asp, Gln, Glu, Gly, Ile, Lys, Met, Pro, Ser, or Trp;

[0339] X₈ is Arg, Glu, Gly, Lys, Phe, Ser, Trp, or Tyr (preferably Ser); or

[0340] (K) Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-Cys (SEQ ID NO: 11), wherein

[0341] X₂ is Asp, Gln, His, Ile, Leu, Lys, Met, Phe, or Thr;

[0342] X₃ is His, Ile, Leu, Met, Phe, Pro, Trp, or Tyr;

[0343] X₄ is Asp, His, Leu, or Ser (preferably Asp);

[0344] X₅ is Ala, Arg, Asp, Glu, Leu, Phe, Pro, or Thr (preferably Glu or Pro);

[0345] X₆ is Ala, Arg, Asn, or Leu (preferably Leu);

[0346] X₇ is Ile, Leu, Met, Pro, Ser, or Thr (preferably Thr);

[0347] X₈ is Ala, Arg, Asn, Gly, His, Lys, Ser, or Tyr;

[0348] X₉ is Ala, Arg, Asn, Gln, Leu, Met, Ser, Trp, Tyr, or Val; or

[0349] (L) Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys (SEQ ID NO: 12), wherein

[0350] X₂ is Arg, Asn, Gln, Glu, His, Leu, Phe, Pro, Trp, Tyr, or Val (preferably Trp, Tyr, or Val);

[0351] X₃ is Arg, Asp, Gln, Gly, Ile, Lys, Phe, Thr, Trp or Tyr (preferably Asp);

[0352] X₄ is Ala, Arg, Asp, Glu, Gly, Leu, Ser, or Tyr (preferably Asp);

[0353] X₅ is Asp, Gln, Glu, Leu, Met, Phe, Pro, Ser, or Tyr (preferably Leu);

[0354] X₆ is Asp, Leu, Pro, Thr, or Val (preferably Leu or Thr);

[0355] X₇ is Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp or Tyr (preferably Lys or Thr);

[0356] X₈ is Ala, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, or Thr (preferably Arg or Leu);

[0357] X₉ is Ala, Asn, Gln, Gly, Leu, Lys, Phe, Pro, Thr, Trp, or Tyr (preferably Thr or Trp);

[0358] X₁₀ is Ala, Arg, Gln, His, Lys, Met, Phe, Pro, Thr, Trp, or Tyr (preferably Met or Phe);

[0359] X₁₁ is Arg, Gln, Glu, Gly, His, Leu, Met, Phe, Pro, Ser, Thr, Tyr, or Val (preferably Val); wherein said polypeptides bind BLyS and/or BLyS-like polypeptides.

[0360] In additional preferred embodiments of the present invention, BLyS binding polypeptides comprise the following amino acid sequence M (SEQ ID NO: 447):

[0361] (M) Ala-X₂-X₃-X₄-Asp-X₆-Leu-Thr-X₉-Leu-X₁₁-X₁₂-X₁₃-X₁₄ (SEQ ID NO: 447), wherein

[0362] X₂ is Asn, Ser, Tyr, Asp, Phe, Ile, Gln, His, Pro, Lys, Leu, Met, Thr, Val, Glu, Ala, Gly, Cys, or Trp (i.e., any amino acid except Arg; preferably Asn);

[0363] X₃ is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly, or Ser (preferably Trp);

[0364] X₄ is Tyr, Phe, Glu, Cys, Asn (preferably Tyr);

[0365] X₆ is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala (preferably Pro or Ser);

[0366] X₉ is Lys, Asn, Gln, Gly, or Arg (preferably Lys);

[0367] X₁₁ is Trp, Ser, Thr, Arg, Cys, Tyr, or Lys (preferably Trp);

[0368] X₁₂ is Leu, Phe, Val, Ile, or His (preferably Leu);

[0369] X₁₃ is Pro, Leu, His, Ser, Arg, Asn, Gln, Thr, Val, Ala, Cys, Ile, Phe, or Tyr (i.e., not Asp, Glu, Gly, Lys, Met, or Trp; preferably Pro); and

[0370] X₁₄ is Asp, Glu, Asn, Val, His, Gln, Arg, Gly, Ser, Tyr, Ala, Cys, Lys, Ile, Thr or Leu (i.e., not Phe, Met, Pro, or Trp; preferably Asp, Val or Glu).

[0371] Preferred embodiments are polypeptides comprising a core sequence of the formula N:

[0372] (N) X₁-X₂-Asp-X₄-Leu-Thr-X₇-Leu-X₉-X₁₀ (SEQ ID NO: 448), wherein

[0373] X₁ is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly, or Ser (preferably Trp);

[0374] X₂ is Tyr, Phe, Glu, Cys, Asn (preferably Tyr);

[0375] X₄ is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala (preferably Pro or Ser);

[0376] X₇ is Lys, Asn, Gln, Gly, or Arg (preferably Lys);

[0377] X₉ is Trp, Ser, Thr, Arg, Cys, Tyr, or Lys (preferably Trp); and

[0378] X₁₀ is Leu, Phe, Val, Ile, or His (preferably Leu).

[0379] Especially preferred BLyS binding polypeptides according to the present invention comprise the core peptide Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu (SEQ ID NO: 436).

[0380] The BLyS binding polypeptides described above may have additional amino acids attached at either or both of the N- and C-terminal ends.

[0381] BLyS binding polypeptide molecules of the invention may also have an amino terminal (N-terminal) capping or functional group, such as an acetyl group, which, for example, blocks the amino terminal amino group from undesirable reactions or is useful in linking the BLyS binding polypeptide to another molecule, matrix, resin, or solid support. BLyS binding polypeptides of the invention may also have a carboxy terminal (C-terminal) capping or functional group, such as an amide group, which, for example, blocks the C-terminal carboxyl group from undesirable reactions or provides a functional group useful in conjugating the binding polypeptide to other molecules, matrices, resins, or solid supports. Preferably, the N- and/or C-terminal capping groups are polypeptide linker molecules. An especially preferred C-terminal linker molecule that is useful for immobilizing a BLyS binding polypeptide of the invention to a solid support or chromatographic matrix material comprises the amino acid sequence Pro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ ID NO: 13). Another useful C-terminal linker, e.g., for fluoresceinating peptides, is Gly-Gly-Lys (see Table 15).

[0382] The invention also encompasses, BLyS binding polypeptides that have been modified, for example, to increase or decrease the stability of the molecule, while retaining the ability to bind BLyS and/or BLyS-like polypeptides. An example of a modified BLyS binding polypeptide of the invention is a polypeptide in which one of two cysteine residues is substituted with a non-naturally occurring amino acid that is capable of condensing with the remaining cysteine side chain to form a stable thioether bridge, thereby generating a cyclic BLyS binding polypeptide. Such cyclic thioether molecules of synthetic peptides may be routinely generated using techniques known in the art and described, e.g., in PCT publication WO 97/46251, incorporated herein by reference.

[0383] In another embodiment, the invention provides BLyS binding polypeptides of the invention attached, coupled, linked or adhered to a matrix or resin or solid support. Techniques for attaching linking or adhering polypeptides to matrices, resins and solid supports are well known in the art. Suitable matrices, resins or solid supports for these materials may be any composition known in the art to which binding polypeptides are commonly attached, coupled, linked, or adhered, including but not limited to, a chromatographic resin or matrix, such as SEPHAROSE-4 FF agarose beads, the wall or floor of a well in a plastic microtiter dish, such as used in an enzyme-liked immunosorbent assay (ELISA), or a silica based biochip. Materials useful as solid supports on which to immobilize binding polypeptides of the invention include, but are not limited to, polyacrylamide, agarose, silica, nitrocellulose, paper, plastic, nylon, metal, and combinations thereof. A BLyS binding polypeptide of the invention may be immobilized on a matrix, resin or solid support material by a non-covalent association or by covalent bonding, using techniques known in the art. Preferably, a BLyS binding polypeptide of the invention is immobilized on SEPHAROSE-4 FF agarose chromatographic material. More preferably, a BLyS binding polypeptide of the invention is coupled to a chromatography material such as SEPHAROSE-4FF (agarose). In an even more preferred embodiment, a BLyS binding polypeptide of the invention is coupled to a chromatography material using a linker molecule. A preferred linker molecule according to the present invention is a polypeptide comprising the amino acid sequence Pro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ ID NO: 13). Most preferably, the affinity chromatography material of the invention comprises a BLyS binding polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 163-172, which is linked to a chromatography material by a polypeptide linker molecule having the amino acid sequence Pro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ ID NO: 13). BLyS binding polypeptides of the invention attached, coupled, linked or adhered to a matrix or resin or solid support are useful for methods of detecting, isolating and purifying BLyS and/or BLyS like polypeptides, particularly for purification of BLyS and/or BLyS like polypeptides by affinity chromatography.

[0384] In certain preferred embodiments, the BLyS binding polypeptides of the present invention or phage displaying such binding polypeptides, irreversibly bind the BLyS protein in its native, soluble trimeric form.

[0385] In certain preferred embodiments, the BLyS binding polypeptides of the present invention or phage displaying such binding polypeptides, reversibly bind the BLyS protein in its native, soluble trimeric form.

[0386] BLyS binding polypeptides of the invention bind BLyS target protein with high affinity. In specific embodiments, BLyS binding polypeptides of the invention bind BLyS target proteins with a dissociation constant or K_(D) of less than or equal to 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, or 10⁻⁵ M. More preferably, BLyS binding polypeptides of the invention bind BLyS target proteins with a dissociation constant or K_(D) less than or equal to 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, or 10⁻⁸ M. Even more preferably, BLyS binding polypeptides of the invention bind BLyS target proteins with a dissociation constant or K_(D) less than or equal to 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

[0387] In certain preferred embodiments, BLyS binding polypeptides of the invention reversibly bind BLyS and/or BLyS-like polypeptides and release bound BLyS protein in an active form, preferably in the native soluble trimeric form, under specific release conditions. In specific embodiments, BLyS binding polypeptides of the invention bind BLyS target proteins with off-rates or k_(off) greater than or equal to 10⁻¹⁰ s⁻¹, 5×10⁻⁹ s⁻¹, 10⁻⁹ s⁻¹, 5×10⁻⁸ s⁻¹, 10⁻⁸ s⁻¹, 5×10⁻⁷ s⁻¹, 10⁻⁷ s⁻¹, 5×10⁻⁶ s⁻¹, 10⁻⁶ s⁻¹, 5×10⁻⁵ s⁻¹, 10⁻⁵ s⁻¹, 5×10⁻⁴ s⁻¹, 10⁻⁴ s⁻¹, 5×10⁻³ s⁻¹, 10⁻³ s⁻¹, 5×10⁻² s³¹ ¹, 10⁻² s⁻¹, 5×10⁻¹ s⁻¹, or 10⁻¹ s⁻¹.

[0388] Binding experiments to determine K_(D) and off-rates can be performed in a number of conditions including, but not limited to, [pH 6.0, 0.01% Tween 20], [pH 6.0, 0.1% gelatin], [pH5.0, 0.01% Tween 20], [pH9.0, 0.1% Tween 20], [pH6.0, 15% ethylene glycol, 0.01% Tween20], [pH5.0, 15% ethylene glycol, 0.01% Tween 20], and [pH9.0, 15% ethylene glycol, 0.01% Tween 20] The buffers in which to make these solutions can readily be determined by one of skill in the art, and depend largely on the desired pH of the final solution. Low pH solutions (<pH 5.5) can be made, for example, in citrate buffer, glycine-HCl buffer, or in succinic acid buffer. High pH solutions can be made, for example, in Tris-HCl, phosphate buffers, or sodium bicarbonate buffers. A number of conditions may be used to determine K_(D) and off-rates for the purpose of determining, for example, optimal pH and/or salt concentrations.

[0389] In certain embodiments, BLyS binding polypeptides of the invention reversibly bind BLyS and/or BLyS-like polypeptides, preferably in the native soluble, trimeric form.

[0390] In preferred embodiments, BLyS binding polypeptides of the invention reversibly bind only the native soluble, trimeric form of BLyS.

[0391] In certain embodiments, BLyS binding polypeptides of the invention irreversibly bind BLyS and/or BLyS-like polypeptides, preferably in the native soluble, trimeric form.

[0392] In preferred embodiments, BLyS binding polypeptides of the invention irreversibly bind only the native soluble, trimeric form of BLyS.

[0393] In some screening or assay procedures, it is possible and more convenient to use recombinant bacteriophage that display a particular BLyS binding polypeptide instead of using isolated BLyS binding polypeptide. Such procedures include phage-based ELISA protocols and immobilization of phage displaying a binding polypeptide to chromatographic materials. Such screening assays and procedures are routine in the art and may be readily adapted for procedures using the recombinant bacteriophage of the present invention.

[0394] The invention also encompasses BLyS binding polypeptides that competitively inhibit the binding of a BLyS binding polypeptide disclosed herein (e.g., a polypeptide having the amino acid sequence of SEQ ID NOS: 163-168) for binding to BLyS. Competitive inhibition can be determined by any suitable method known in the art, for example, using the competitive binding assays described herein. In preferred embodiments, the polypeptide competitively inhibits the binding of a BLyS binding polypeptide disclosed herein (e.g., a polypeptide having the amino acid sequence of SEQ ID NOS: 163-168) to BLyS by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%. In a more preferred embodiment, the BLyS binding polypeptide competitively inhibits the binding of a BLyS binding polypeptide disclosed herein (e.g., a polypeptide having the amino acid sequence of SEQ ID NOS: 163-168) to the native soluble trimeric form of BLyS, by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.

[0395] In a further embodiment, the present invention encompasses a composition of matter comprising isolated nucleic acids, preferably DNA, encoding a BLyS binding polypeptide of the invention. In a specific embodiment, nucleic acid molecules of the invention encode a BLyS binding polypeptide of the invention as provided in SEQ ID NOs: 1-12, 20-172, and 186-444. In additional embodiments, nucleic acid molecules of the invention encode a polypeptide variant or fragment of a polypeptide having an amino acid sequence of SEQ ID NOs: 1-12, 20-172, and 186-444. In a further additional embodiment, nucleic acid molecules of the invention encode a BLyS binding polypeptide, the complementary strand of which nucleic acid hybridizes to a polynucleotide sequence encoding a polypeptide described in Tables 1-8 and in Examples 2 and 5 (SEQ ID NOs: 1-12, 20-172, and 186-444), under stringent conditions, e.g., hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate (SSC) at about 450 C followed by one or more washes in 0.2×SSC/0.1% SDS at about 50-65° C., under highly stringent conditions, e.g., hybridization to filter-bound nucleic acid in 6×SSC at about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C., or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel et al., eds. , 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3).

[0396] The present invention also relates to recombinant vectors that include the isolated nucleic acid molecules encoding the BLyS binding polypeptides of the present invention (as well as fragments and variants thereof), and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells. The invention further provides for the use of such recombinant vectors in the production of BLyS binding polypeptides by recombinant techniques.

[0397] The BLyS binding polypeptides, nucleic acids, transformed host cells, and genetically engineered viruses and phage of the invention (e.g., recombinant phage), have uses that include, but are not limited to, the detection, isolation, and purification of BLyS.

[0398] In another embodiment of the invention, recombinant bacteriophage displaying BLyS binding polypeptides on their surfaces are also provided. Such phage may be routinely generated using techniques known in the art and are useful, for example, as screening reagents and reagents for detecting BLyS.

[0399] Production and Modification of BLyS Binding Polypeptides

[0400] BLyS binding polypeptides of the invention may be produced by chemical synthesis, semi-synthetic methods, and recombinant DNA methodologies known in the art.

[0401] In certain embodiments, BLyS binding polypeptides of the present invention are produced by chemical or semi-synthetic methodologies known in the art (see, Kelley et al. in Genetic Engineering Principles and Methods, Setlow, J. K., ed. (Plenum Press, NY., 1990), vol. 12, pp. 1-19; Stewart et al., Solid-Phase Peptide Synthesis, W. H. Freeman Co., San Francisco, 1989). One advantage of these methodologies is that they allow for the incorporation of non-natural amino acid residues into the sequence of the BLyS binding polypeptide.

[0402] In preferred embodiments, BLyS binding polypeptides of the invention are chemically synthesized (see, e.g., Merrifield, J. Am. Chem. Soc., 85: 2149 (1963); Houghten, Proc. Natl. Acad. Sci. USA, 82: 5132 (1985)). For example, polypeptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (see, e.g., Creighton, Proteins: Structures and Molecular Properties (W. H. Freeman and Co., N.Y., 1983), pp. 50-60). BLyS binding polypeptides can also be synthesized by use of a peptide synthesizer. The composition of the synthetic polypeptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, Proteins: Structures and Molecular Properties (W. H. Freeman and Co., N.Y., 1983), pp. 34-49). Furthermore, if desired, BLyS binding polypeptides of the invention may contain non-classical amino acids or chemical amino acid analogs, which can routinely be introduced during chemical synthesis as a substitution or addition into the BLyS binding polypeptides of the invention. Non-classical amino acids include, but are not-limited to, the D-isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid (4Abu), 2-aminobutyric acid (Abu), 6-aminohexanoic acid (epsilon-Ahx), 2-aminoisobutyric acid (Aib), 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine (bAla), fluoro-amino acids, designer amino acids such as beta-methyl amino acids, Calpha-methyl amino acids, Nalpha-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

[0403] Solid phase peptide synthesis begins at the carboxy (C) terminus of the putative polypeptide by coupling a protected amino acid to a suitable resin, which reacts with the carboxyl group of the C-terminal amino acid to form a bond that is readily cleaved later, for example, a halomethyl resin such as chloromethyl resin, bromomethyl resin, hydroxymethyl resin, aminomethyl resin, benzhydrylamine resin, or t-alkyloxycarbonyl-hydrazide resin. After removal of the α-amino protecting group with, for example, trifluoroacetic acid (TFA) in methylene chloride and neutralization with, for example TEA, the next cycle in the synthesis is ready to proceed. The remaining α-amino and, if necessary, side-chain-protected amino acids are then coupled sequentially in the desired order by condensation to obtain an intermediate compound connected to the resin. Alternatively, some amino acids may be coupled to one another forming an oligopeptide prior to addition to the growing solid phase polypeptide chain.

[0404] The condensation between two amino acids, or an amino acid and a peptide, or a peptide and a peptide can be carried out according to condensation methods known in the art, including but not limited to, the azide method, mixed acid anhydride method, DCC (dicyclohexylcarbodiimide) method, active ester method (p-nitrophenyl ester method, BOP [benzotriazole-1-yl-oxy-tris (dimethylamino) phosphonium hexafluorophosphate] method, N-hydroxysuccinic acid imido ester method), and Woodward reagent K method.

[0405] Common to chemical synthesis of peptides is the protection or capping (blocking) of the reactive side chain groups of the various amino acid residues with suitable protecting or capping groups at that site until the group is ultimately removed after the polypeptide chain has been completely assembled. Also common is the protection or capping of the α-amino group on an amino acid or a fragment while that entity reacts at the carboxyl group followed by the selective removal of the α-amino-protecting group to allow subsequent reaction to take place at that location. Accordingly, during synthesis, intermediate compounds are produced which includes each of the amino acid residues located in the desired sequence in the peptide chain with various of these residues having side-chain protecting or capping groups. These protecting or capping groups on amino acid side chains are then removed substantially at the same time so as to produce the desired resultant product following purification.

[0406] The typical protective, capping, or blocking groups for α- and ε-amino side chain groups found in amino acids are exemplified by benzyloxycarbonyl (Z), isonicotinyloxycarbonyl (iNOC), O-chlorobenzyloxycarbonyl [Z(NO₂)], p-methoxybenzyloxycarbonyl [Z(OMe)], t-butoxycarbonyl (Boc), t-amyioxycarbonyl (Aoc), isobornyloxycarbonyl, adamatyloxycarbonyl, 2-(4-biphenyl)-2-propyloxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl (Fmoc), methylsulfonyiethoxycarbonyl (Msc), trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulphenyl (NPS), diphenylphosphinothioyl (Ppt), dimethylophosphinothioyl (Mpt), and the like.

[0407] Protective, capping, or blocking groups for the carboxyl group of amino acids include, for example, benzyl ester (OBzl), cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb), t-butyl ester (Obut), 4-pyridylmethyl ester (OPic), and the like. It is usually also desirable that side chain groups of specific amino acids such as arginine, cysteine, and serine, are protected by a suitable protective group as occasion demands. For example, the guanidino group in arginine may be protected with nitro, p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzenesulfonyl, 4-methoxy-2,6-dimethylbenzenesulfonyl (Mds), 1,3,5-trimethylphenylsulfonyl (Mts), and the like. The thiol group in cysteine may be protected with p-methoxybenzyl, triphenylmethyl, acetylaminomethyl ethylcarbamoyl, 4-methylbenzyl, 2,4,6-trimethy-benzyl (Tmb), etc., and the hydroxyl group in the serine can be protected with benzyl, t-butyl, acetyl, tetrahydropyranyl, etc.

[0408] After the desired amino acid sequence has been completed, the intermediate polypeptide is removed from the resin support by treatment with a reagent, such as liquid HF and one or more thio-containing scavengers, which cleaves the peptide molecule from the resin and all the remaining side-chain protecting groups. Following HF cleavage, the protein sequence is washed with ether, transferred to a large volume of dilute acetic acid, and stirred at pH adjusted to about 8.0 with ammonium hydroxide. Upon pH adjustment, the polypeptide takes its desired conformational arrangement.

[0409] By way of example but not by way of limitation, polypeptides of the invention can be chemically synthesized and purified as follows: Peptides can be synthesized by employing the N-alpha-9-fluorenylmethyloxycarbonyl or Fmoc solid phase peptide synthesis chemistry using a Rainin Symphony Multiplex Peptide Synthesizer. The standard cycle used for coupling of an amino acid to the peptide-resin growing chain generally includes: (1) washing the peptide-resin three times for 30 seconds with N,N-dimethylformamide (DMF); (2) removing the Fmoc protective group on the amino terminus by deprotection to with 20% piperdine in DMF by two washes for 15 minutes each, during which process mixing is effected by bubbling nitrogen through the reaction vessel for one second every 10 seconds to prevent peptide-resin settling; (3) washing the peptide-resin three times for 30 seconds with DMF; (4) coupling the amino acid to the peptide resin by addition of equal volumes of a 250 mM solution of the Fmoc derivative of the appropriate amino acid and an activator mix consisting or 400 mM N-methylmorpholine and 250 mM (2-(1H-benzotriazol-1-4))-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) in DMF; (5) allowing the solution to mix for 45 minutes; and (6) washing the peptide-resin three times for 30 seconds of DMF. This cycle can be repeated as necessary with the appropriate amino acids in sequence to produce the desired peptide. Exceptions to this cycle program are amino acid couplings predicted to be difficult by nature of their hydrophobicity or predicted inclusion within a helical formation during synthesis. For these situations, the above cycle can be modified by repeating step 4 a second time immediately upon completion of the first 45 minute coupling step to “double couple” the amino acid of interest. Additionally, in the first coupling step in peptide synthesis, the resin can be allowed to swell for more efficient coupling by increasing the time of mixing in the initial DMF washes to three 15 minute washes rather than three 30 second washes.

[0410] After peptide synthesis, the peptide can be cleaved from the resin as follows: (1) washing the peptide-resin three times for 30 seconds with DMF; (2) removing the Fmoc protective group on the amino terminus by washing two times for 15 minutes it 20% piperdine in DMF; (3) washing the peptide-resin three times for 30 seconds with DMF; and (4) mixing a cleavage cocktail consisting of 95% trifluoroacetic acid (TFA), 2.4% water, 2.4% phenol, and 0.2% triisopropysilane with the peptide-resin for two hours, then filtering the peptide in the cleavage cocktail away from the resin, and precipitating the peptide out of solution by addition of two volumes of ethyl ether. Specifically, to isolate the peptide, the ether-peptide solution can be allowed to sit at −20° C. for 20 minutes, then centrifuged at 6,000×G for 5 minutes to pellet the peptide, and the peptide can be washed three times with ethyl ether to remove residual cleavage cocktail ingredients. The final peptide product can be purified by reversed phase high pressure liquid chromatography (RP-HPLC) with the primary solvent consisting of 0.1% TFA and the eluting buffer consisting of 80% acetonitrile and 0.1% TFA. The purified peptide can then be lyophilized to a powder.

[0411] In other specific embodiments, branched versions of the BLyS binding polypeptides described herein are provided, e.g., by substituting one or more amino acids within the BLyS binding polypeptide sequence with an amino acid or amino acid analog with a free side chain capable of forming a peptide bond with one or more amino acids (and thus capable of forming a “branch”).

[0412] Branched peptides may be prepared by any method known in the art for covalently linking any naturally occurring or synthetic amino acid to any naturally occurring or synthetic t amino acid in a peptide chain which has a side chain group able to react with the amino or carboxyl group on the amino acids so as to become covalently attached to the peptide chain. In particular, amino acids with a free amino side chain group, such as, but not limited to, diaminobutyric acid, lysine, arginine, ornithine, diaminopropionic acid and citrulline, can be incorporated into a peptide so that an amino acid can form a branch therewith, for example, by forming a peptide bond to the free amino side group, from that residue. Alternatively, amino acids with a free carboxyl side chain group, such as, but not limited to, glutamic acid, aspartic acid and homocitrulline, can be incorporated into the peptide so that an amino acid can form a branch therewith, for example, by forming a peptide bond to the free carboxyl side group, from that residue. The amino acid forming the branch can be linked to a side chain group of an amino acid in the peptide chain by any type of covalent bond, including, but not limited to, peptide bonds, ester bonds and disulfide bonds. In a specific embodiment, amino acids, such as those described above, that are capable of forming a branch point, are substituted for BLyS binding polypeptide residues within a peptide including a BLyS binding polypeptide sequence.

[0413] Branched peptides can be prepared by any method known in the art. For example, but not by way of limitation, branched peptides can be prepared as follows: (1) the amino acid to be branched from the main peptide chain can be purchased as an N-alpha-tert-butyloxycarbonyl (Boc) protected amino acid pentafluorophenyl (Opfp) ester and the residue within the main chain to which this branched amino acid will be attached can be an N-Fmoc-alpha-gamma-diaminobutyric acid; (2) the coupling of the Boc protected amino acid to diaminobutyric acid can be achieved by adding 5 grams of each precursor to a flask containing 150 ml DMF, along with 2.25 ml pyridine and 50 mg dimethylaminopyridine and allowing the solution to mix for 24 hours; (3) the peptide can then be extracted from the 150 ml coupling reaction by mixing the reaction with 400 ml dichlormethane (DCM) and 200 ml 0.12N HCl in a 1 liter separatory funnel, and allowing the phases to separate, saving the bottom aqueous layer and re-extracting the top layer two more times with 200 ml 0.12N HCl; (4) the solution containing the peptide can be dehydrated by adding 2-5 grams magnesium sulfate, filtering out the magnesium sulfate, and evaporating the remaining solution to a volume of about 2-5 ml; (5) the dipeptide can then be precipitated by addition of ethyl acetate and then 2 volumes of hexanes and then collected by filtration and washed two times with cold hexanes; and (6) the resulting filtrate can be lyophilized to achieve a light powder form of the desired dipeptide. Branched peptides prepared by this method will have a substitution of diaminobutyric acid at the amino acid position which is branched. Branched peptides containing an amino acid or amino acid analog substitution other than diaminobutyric acid can be prepared analogously to the procedure described above, using the N-Fmoc coupled form of the amino acid or amino acid analog.

[0414] In a preferred embodiment, the BLyS binding polypeptide of the invention is a cyclic peptide. Cyclization can be, for example, but not by way of limitation, via a disulfide bond between two cysteine residues or via an amide linkage. For example, but not by way of limitation, disulfide bridge formation can be achieved by (1) dissolving the purified peptide at a concentration of between 0.1-0.5 mg/ml in 0.01 M ammonium acetate, pH 7.5; (2) adding to the dissolved peptide 0.01 M potassium ferricyanide dropwise until the solution appears pale yellow in color and allowing this solution to mix for 24 hours; (3) concentrating the cyclized peptide to 5-10 ml of solution, repurifying the peptide by reverse phase-high pressure liquid chromatography (RP-HPLC) and finally lyophilizing the peptide. In a specific embodiment, in which the peptide does not contain two appropriately situated cysteine residues, cysteine residues can be introduced at the amino-terminus and/or carboxy-terminus and/or internally such that the peptide to be cyclized contains two cysteine residues spaced such that the residues can form a disulfide bridge. Alternatively, a cyclic peptide can be obtained by generating an amide linkage using, for example but not limited to, the following protocol: An allyl protected amino acid, such as aspartate, glutamate, asparagine or glutamine, can be incorporated into the peptide as the first amino acid, and then the remaining amino acids are coupled on. The allyl protective group can U be removed by a two hour mixing of the peptide-resin with a solution of tetrakistriphenylphosphine palladium (0) in a solution of chloroform containing 5% acetic acid and 2.5% N-methylmorpholine. The peptide resin can be washed three times with 0.5% N,N-diisopropylethylamine (DIEA) and 0.5% sodium diethyldithiocabamate in DMF. The amino terminal Fmoc group on the peptide chain can be removed by two incubations for 15 minutes each in 20% piperdine in DMF, and washed three times with DMF for 30 seconds each. The activator mix, N-methylmorpholine and HBTU in DMF, can be brought onto the column and allowed to couple the free amino terminal end to the carboxyl group generated by removal of the allyl group to cyclize the peptide. The peptide can be cleaved from the resin as described in the general description of chemical peptide synthesis above and the peptide purified by reverse phase-high pressure liquid chromatography (RP-HPLC). In a specific embodiment, in which the peptide to be cyclized does not contain an allyl protected amino acid, an allyl protected amino acid can be introduced into the sequence of the peptide, at the amino-terminus, carboxy-terminus or internally, such that the peptide can be cyclized.

[0415] In addition, according to certain embodiments, it is preferable that the BLyS binding polypeptides of the invention are produced having or retaining an amino terminal (N-terminal) and/or a carboxy terminal (C-terminal) capping group, which may protect the N-terminal or C-terminal amino acid from undesirable chemical reactions during use or which may permit further conjugations or manipulations of the binding polypeptide, for example, in conjugating the binding polypeptide to a chromatographic support resin or matrix or to another peptide to tether the binding polypeptide to a resin or support. Such N-terminal and C-terminal groups may also be used to label or tag the binding polypeptide to detect bound complexes or to locate the binding polypeptide (whether bound or unbound to a BLyS target protein) for example, at some point in a separation procedure. Accordingly, a BLyS binding polypeptide of the invention synthesized in its final form for use in a detection or separation procedure may contain an N-terminal and/or a C-terminal capping group. A particularly preferred N-terminal capping group, which may be present or retained in binding polypeptides of the invention, is an acetyl group (Ac). A particularly preferred C-terminal capping group, which may be present or retained in binding polypeptides of the invention, is an amide group. In a further preferred embodiment, the BLyS binding polypeptides of the invention have an acetyl group as an N-terminal capping group and an amide group as a C terminal capping group.

[0416] The BLyS binding polypeptides of the invention may also be prepared commercially by companies providing polypeptide synthesis as a service (e.g., BACHEM Bioscience, Inc., King of Prussia, Pa.; Quality Controlled Biochemicals, Inc., Hopkinton, Mass.).

[0417] The nucleic acid sequence encoding a BLyS binding polypeptide of the invention can be produced and isolated using well-known techniques in the art. In one example, nucleic acids encoding the BLyS binding polypeptides of the invention are chemically synthesized based on knowledge of the amino acid sequence of the BLyS binding polypeptide (preferably the sequence is codon optimized to the host system in which the polypeptide will be expressed). In another example, nucleic acids encoding a BLyS binding polypeptide are obtained by screening an expression library (e.g., a phage display library) to identify phage expressing BLyS binding polypeptides, and isolating BLyS binding polypeptide encoding nucleic acid sequences from the identified library member (e.g., via polymerase chain reaction methodology using primers flanking the polypeptide encoding sequences).

[0418] The present invention also relates to vectors which include nucleic acid sequences encoding the BLyS binding polypeptides of the invention, host cells which are genetically engineered with the recombinant vectors, or which are otherwise engineered to produce the polypeptides of the invention, and the production of BLyS binding polypeptides, or fragments thereof, by recombinant, chemical or synthetic techniques.

[0419] Thus, according to the present invention, BLyS binding polypeptidess can also be obtained by recombinant expression techniques. (See, e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Glover, D. M. (ed.), (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989); DNA Cloning: A Practical Approach (MRL Press, Ltd., Oxford, U.K., 1985), Vols. I, II.

[0420] To produce a recombinant BLyS binding polypeptide, a nucleic acid sequence encoding the BLyS binding polypeptide is operatively linked to a promoter such that the BLyS binding polypeptide is produced from said sequence. For example, a vector can be introduced into a cell, within which cell the vector or a portion thereof is expressed, producing the BLyS binding polypeptides. In a preferred embodiment, the nucleic acid is DNA if the source of RNA polymerase is DNA-directed RNA polymerase, but the nucleic acid may also be RNA if the source of polymerase is RNA-directed RNA polymerase or if reverse transcriptase is present in the cell or provided to produce DNA from the RNA. Such a vector can remain episomal or, become chromosomally integrated, as long as it can be transcribed to produce the desired RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be bacteriophage, plasmid, viral, retroviral, or others known in the art, used for replication and expression in bacterial, fungal, plant, insect or mammalian cells. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells. Introduction of the vector construct into the host cell can be effected by techniques known in the art which include, but are not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986).

[0421] Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, for example, stabilization or simplified purification of expressed recombinant product.

[0422] In one embodiment, the nucleic acid encoding a BLyS binding polypeptide of the invention is operatively associated with an appropriate heterologous regulatory element (e.g., promoter or enhancer), such as, the phage lambda PL promoter, the E. coli lac, trp, phoA, and tac promoters, the SV40 early and late promoters, and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan.

[0423] As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, NSO and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

[0424] The host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. The host strain may be chosen which modulates the expression of at the inserted nucleic acid sequences encoding the BLyS polypeptides of the invention, or modifies and processes the Blys binding polypeptide in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled. Furthermore, different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed. Selection of appropriate vectors and promoters for expression in a host cell is a well-known procedure and the requisite techniques for expression vector construction, introduction of the vector into the host and expression in the host are routine skills in the art.

[0425] Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will preferably comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice. As a representative, but i nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well-known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA). These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed. Among vectors preferred for use in bacteria are pHE4-5 (ATCC Accession No. 209311) and variations thereof), pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlsbad, Calif.). Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL (available from Pharmacia). Other suitable vectors will be readily apparent to the skilled artisan.

[0426] Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

[0427] Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well known to those skilled in the art.

[0428] In one embodiment, the yeast Pichia pastoris is used to express a BLyS binding polypeptide in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O₂. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O₂. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOX1) is highly active. In the presence of methanol, alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See, Ellis et al., Mol. Cell. Biol., 5:1111-21 (1985); Koutz et al., Yeast, 5:167-77 (1989); Tschopp et al., Nucl. Acids Res., 15:3859-76 (1987). Thus, a heterologous coding sequence, such as, for example, a polynucleotide encoding a BLyS binding polypeptide of the present invention, under the transcriptional regulation of all or part of the AOX1 regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.

[0429] In one example, the plasmid vector pPIC9K is used to express DNA encoding a BLyS binding polypeptide of the invention, as set forth herein, in a Pichea yeast system essentially as described in “Pichia Protocols: Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. (The Humana Press, Totowa, N.J., 1998). This expression vector allows expression and secretion of a BLyS binding polypeptide of the invention by virtue of the strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.

[0430] Many other yeast vectors may be used in place of pPIC9K, such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG as required.

[0431] In one embodiment, high-level expression of a heterologous coding sequence, such as, for example, a nucleic acid encoding a BLyS binding polypeptide of the invention, may be achieved by cloning the heterologous nucleic acid sequence of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.

[0432] Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0433] Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman (Cell, 23:175 (1981)), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, 293, NSO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

[0434] The host cells described herein may be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, cell-free translation systems can also be employed to produce the polypeptides of the invention using RNAs derived from the DNA constructs of the present invention.

[0435] The polypeptides of the invention may be expressed or synthesized in a modified form, such as a fusion protein (comprising the polypeptide joined via a peptide bond to a heterologous protein sequence (of a different protein)), and may include not only secretion signals, but also additional heterologous functional regions. Such a fusion protein can be made by ligating polynucleotides of the invention and the desired nucleic acid sequence encoding the desired amino acid sequence to each other, by methods known in the art, in the proper reading frame, and expressing the fusion protein product by methods known in the art. Alternatively, such a fusion protein can be made by protein synthetic techniques, e.g., by use of a peptide synthesizer. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art. Particular mention is made of the hexa-histidine polypeptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA, 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin“HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell, 37:767 (1984)) and the “flag” tag (DYKDDDDK, (SEQ ID NO: 183) Stratagene, La Jolla, Calif.).

[0436] In one embodiment, nucleic acids encoding a BLyS binding polypeptides of the invention may be fused to the pelB pectate lyase signal sequence to increase the efficiency to expression and purification of such polypeptides in Gram-negative bacteria. See, U.S. Pat. Nos. 5,576,195 and 5,846,818, the contents of which are herein incorporated by reference in their entireties.

[0437] Polypeptides of the present invention include products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.

[0438] The invention encompasses BLyS binding polypeptides which are modified during or after synthesis or translation, e.g., by glycosylation, acetylation, benzylation, phosphorylation, amidation, pegylation, formylation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, ubiquitination, etc. (See, for instance, Creighton, Proteins: Structures and Molecular Properties, 2d Ed. (W. H. Freeman and Co., N.Y., 1992); Postranslational Covalent Modification of Proteins, Johnson, ed. (Academic Press, New York, 1983), pp. 1-12; Seifter et al., Meth. Enzymol., 182:626-646 (1990); Rattan et al., Ann. NY Acad. Sci., 663:48-62 (1992).) In specific embodiments, the peptides are acetylated at the N-terminus and/or amidated at the C-terminus.

[0439] In further embodiments, BLyS binding polypeptides of the invention containing two or more residues that have the potential to interact, such as for example, two cysteine residues in a polypeptide, may be treated under oxidizing conditions or other conditions that promote interaction of these residues (e.g., dislulfide bridge formation).

[0440] Further BLyS binding polypeptide modifications encompassed by the invention include, for example, any of numerous chemical modifications carried out by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.

[0441] Additional post-translational /post-synthesis modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.

[0442] Also provided by the invention are chemically modified derivatives of BLyS binding polypetides of the invention which may provide additional advantages such as increased affinity, decreased off-rate, solubility, stability and in vivo or in vitro circulating time of the polypeptide, or decreased immunogenicity (see, U.S. Pat. No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

[0443] The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any, on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

[0444] As noted above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol., 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides, 18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem., 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.

[0445] The polyethylene glycol molecules (or other chemical moieties) should be attached to the BLyS binding poypeptide with consideration of effects on functional domains of the polypeptide. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol., 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include, for example, lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues, and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. In a preferred embodiment, the polyethylene glycol molecule is attached at an amino group, such as attachment at the N-terminus or to a lysine side chain amino group.

[0446] As suggested above, polyethylene glycol may be attached to polypeptides via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to a polypeptide via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the polypeptide or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the polypeptide.

[0447] One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to polypeptide molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated polypeptide. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated polypeptide molecules. Selective N-terminal modification of proteins may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminus) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

[0448] As indicated above, pegylation of the polypeptides of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the protein either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys., 9:249-304 (1992); Francis et al., Intern. J. of Hematol., 68:1-18 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.

[0449] One system for attaching polyethylene glycol directly to amino acid residues of polypeptides without an intervening linker employs tresylated MPEG, which is produced by the modification of monomethoxy polyethylene glycol (PEG) using tresylchloride (ClSO₂CH₂CF₃). Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the polyeptide. Thus, the invention includes polypeptide-polyethylene glycol conjugates produced by reacting polypeptides of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.

[0450] Polyethylene glycol can also be attached to polypeptides using a number of different intervening linkers. For example, U.S. Pat. No. 5,612,460, the entire disclosure of which is incorporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to polypeptides. Polypeptide-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the polypeptide by a linker can also be produced by reaction of polypeptides with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1′-carbonyldiimidazole, MPEG-2,4,5-trichlorophenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number of additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to polypeptides are described in WO 98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated BLyS binding polypeptide products produced using the reaction chemistries set out herein are included within the scope of the invention.

[0451] The number of polyethylene glycol moieties attached to each polypeptide of the invention (i.e., the degree of substitution) may also vary. For example, the pegylated polypeptides of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution may range within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per polypeptide molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys., 9:249-304 (1992).

[0452] BLyS Binding Polypeptide Multimers, Conjugates and Fusions

[0453] The present invention encompasses multivalent BLyS binding polypeptides. BLyS binding polypeptides may be monomeric, dimeric, trimeric, or higher-order multimers. In a preferred embodiment multivalent BLyS binding polypeptides are homotrimeric. In another preferred embodiment a homotrimeric BLyS binding polypeptide binds a single homotrimeric BLyS.

[0454] In another preferred embodiment, monomeric or multimeric BLyS binding polypeptides are conjugated with another polypeptide or other chemical compound. For example, BLyS binding polypeptide(s) may be conjugated to a radioactive or other toxic compound so as to target and destroy cells expressing BLyS.

[0455] The present invention also encompasses heteromeric multimers comprised of one or more BLyS binding polypeptides and one or more non-BLyS binding polypeptides or other chemical moieties. Such heteromeric multimers may be monomeric, dimeric, trimeric, tetrameric, pentameric, or higher-order multimers. Heteromeric BLyS binding multimers may be used to target, bind, inhibit, and/or activate responses in cells expressing BLyS and receptors for the heterologous, non-BLyS binding polypeptide or other chemical moiety. Such activated responses may include, for example, apoptosis or other biologically and chemically mediated forms of cell destruction. Heteromeric BLyS binding multimers may also be used to target BLyS expressing cells so as to introduce a desired molecule or compound to the cells. For example, a heteromeric BLyS binding multimer may be conjugated with a radioactive or otherwise toxic compound so as to kill BLyS expressing cells. As an alternative example, a heteromeric BLyS binding and Adenovirus-binding multimer could be used to specifically target and introduce adenovirus-mediated gene therapeutics into BLyS expressing cells.

[0456] BLyS binding polypeptide multimers may be fused or conjugated as homopolymers and heteropolymers using methods known in the art. In a preferred embodiment BLyS binding polypeptides are linked as homomultimers wherein the linker or linkers provide sufficient length and flexibility such that each BLyS binding polypeptide may simultaneously bind an individual BLyS molecule. In another preferred embodiment BLyS binding polypeptides are linked as heteromultimers wherein the linker or linkers provide sufficient length and flexibility such that each BLyS binding polypeptide may simultaneously bind individual BLyS molecules and the heterologous polypeptide or chemical moiety may simultaneously bind to its target. Numerous examples of suitable linker molecules are known in the art. (See, for example, Todorovska et al., J. Immunol. Methods, 248(1-2):47-66 (2001); Mehvar, J. Control Release, 69(1):1-25 (2000); Francis et. al., Int. J. Hematol., 68(1):1-18 (1998).) In specific embodiments, the linker is a member selected from the group consisting of: (a) a peptide linker; (b) a glutamate linker; and (c) a polyethylene glycol linker. The length of linkers to be used according to the methods of the invention may routinely be determined using techniques known in the art. In specific embodiments, the linker is 5-60 angstroms in length. In other embodiments, the linker is 10-50, 10-40, 10-30, or 10-20 angstroms in length. In further embodiments, the linker is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 angstroms in length. In this context “about” includes the recited length, and/or lengths that are larger or smaller by several (5, 4, 3, 2, or 1) angstroms. In other embodiments, the linker is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 angstroms in length.

[0457] In a preferred embodiment, BLyS binding polypeptides may be fused with human serum albumin (HA). See, e.g., U.S. application Ser. No. 09/833,245, filed Apr. 12, 2001, which is hereby incorporated by reference herein. In one embodiment, the albumin fusion protein comprises HA as the N-terminal portion, and a BLyS binding polypeptide as the C-terminal portion. In another embodiment the albumin fusion protein comprise HA as the C-terminal portion, and a BLyS binding polypeptide as the N-terminal portion.

[0458] In other embodiments, the albumin fusion protein has a BLyS binding polypeptide fused to both the N-terminus and the C-terminus of albumin. In one preferred embodiment, the BLyS binding polypeptides fused at the N- and C-termini are the same BLyS binding polypeptides. In another preferred embodiment, the BLyS binding polypeptides fused at the N- and C-termini are different BLyS binding polypeptides. In another preferred embodiment, a BLyS binding polypeptide is fused at either the N- or C-terminus of HA and a different (non-BLyS binding) polypeptide is fused at either the C- or N-terminus, respectively.

[0459] In addition to albumin fusion proteins in which the BLyS binding polypeptide(s) is (are) fused to the N-terminus and/or C-terminus of HA, BLyS binding polypeptide/albumin fusion proteins of the invention may also be produced by inserting the BLyS binding polypeptide into an internal region or regions of HA. For instance, within the protein sequence of the HA molecule a number of loops or turns exist between the end and beginning of α-helices, which are stabilized by disulphide bonds (see FIGS. 9-11 in U.S. application Ser. No. 09/833,245). The loops, as determined from the crystal structure of HA (FIG. 13 of U.S. application Ser. No. 09/833,245) (PDB identifiers 1A06, 1BJ5, 1BKE, 1BM0, 1E7E to 1E7I and 1UOR) for the most part extend away from the body of the molecule. These loops are useful for the insertion, or internal fusion, of therapeutically active peptides (particularly those requiring a secondary structure to be functional) or therapeutic proteins, to essentially generate an albumin molecule with specific biological activity.

[0460] Loops in human albumin structure into which binding polypeptides of the invention may be inserted to generate albumin fusion proteins of the invention include: Val54-Asn61, Thr76-Asp89, Ala92-Glu100, Gln170-Ala176, His 247-Glu252, Glu 266-Glu277, Glu 280-His288, Ala362-Glu368, Lys439-Pro447, Val462-Lys475, Thr478-Pro486, and Lys560-Thr566. In more preferred embodiments, polypeptides of the invention are inserted into the Val54-Asn61, Gln170-Ala176, and/or Lys560-Thr566 loops of mature human serum albumin (SEQ ID NO: 445).

[0461] In specific embodiments, BLyS binding polypeptides of the invention are attached to macrocyclic chelators useful for conjugating radiometal ions, including but not limited to, ¹¹¹In, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and ¹⁵³Sm, to polypeptides. In a preferred embodiment, the radiometal ion associated with the macrocyclic chelators attached to BLyS binding polypeptides of the invention is ¹¹¹In. In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator attached to BLyS binding polypeptides of the invention is ⁹⁰Y. In specific embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). In other specific embodiments, the DOTA is attached to the BLyS binding polypeptides of the invention via a linker molecule. Examples of linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art—see, for example, DeNardo et al., Clin. Cancer Res., 4(10):2483-90 (1998); Peterson et al., Bioconjug. Chem., 10(4):553-7 (1999); and Zimmerman et al, Nucl. Med. Biol., 26(8):943-50 (1999), which are hereby incorporated by reference in their entirety. In addition, U.S. Pat. Nos. 5,652,361 and 5,756,065, which disclose chelating agents that may be conjugated to antibodies, and methods for making and using them, are hereby incorporated by reference in their entireties. Though U.S. Pat. Nos. 5,652,361 and 5,756,065 focus on conjugating chelating agents to antibodies, one skilled in the art would be readily able to adapt the method disclosed therein in order to conjugate chelating agents to other polypeptides.

[0462] The BLyS binding polypeptides of the invention can be recovered and purified by known methods which include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.

[0463] The BLyS binding polypeptides may also be modified with a detectable label, including, but not limited to, an enzyme, prosthetic group, fluorescent material, luminescent material, bioluminescent material, radioactive material, positron emitting metal, nonradioactive paramagnetic metal ion, and affinity label for detection and isolation of BLyS target. The detectable substance may be coupled or conjugated either directly to the polypeptides of the invention or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include biotin, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include a radioactive metal ion, e.g., alpha-emitters such as, for example, ²¹³Bi, or other radioisotopes such as, for example, iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In), and technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, 175Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁵Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and ¹¹⁷Tin.

[0464] In a specific embodiment, BLyS binding polypeptides of the invention are labeled with biotin.

[0465] Uses of the Binding Polypeptides and Recombinant Bacteriophage of the Invention

[0466] The BLyS binding polypeptides described herein are especially useful to detect, isolate, or remove BLyS target proteins in solutions. Such solutions may be simple dispersions or solutions of BLyS and/or BLyS-like polypeptide in water or aqueous buffer or more complex solutions, such as, a blood and other biological fluids, tissue homogenates cell extracts, or biopsy samples, and cell culture media containing BLyS or BLyS-like polypeptides. Biological fluids include, but are not limited to sera, plasma, lymph, blood, blood fractions urine, synovial fluid, spinal fluid, saliva, and mucous.

[0467] In one embodiment, the present invention provides a method for detecting a BLyS protein and/or a BLyS-like polypeptide in a solution comprising contacting the solution with a BLyS binding polypeptide of the invention and detecting binding of BLyS or BLyS-like polypeptide to the BLyS binding polypeptide. The BLyS binding polypeptide may be either free or immobilized. Preferably, the BLyS binding polypeptide is a polypeptide immobilized on a solid surface or chromatographic material or the well of a plastic microtiter assay dish.

[0468] Another embodiment of the present invention is a method for isolating BLyS protein and/or BLyS-like polypeptide from a solution containing it, comprising:

[0469] (a) contacting the solution with a BLyS binding polypeptide under conditions that permit binding of BLyS and/or BLyS-like polypeptides to BLyS binding polypeptide, and

[0470] (b) recovering the BLyS and/or BLyS-like polypeptides.

[0471] A further embodiment of the present invention is a method for isolating BLyS protein and/or BLyS-like polypeptide from a solution containing it, comprising:

[0472] (a) contacting the solution with a BLyS binding polypeptide under conditions that permit binding of BLyS and/or BLyS-like polypeptides to BLyS binding polypeptide, and

[0473] (b) separating the complex(es) formed by the BLyS binding polypeptide and BLyS and/or BLyS-like polypeptides from other components of the solution.

[0474] Preferably such method also includes the further steps of:

[0475] (c) dissociating the BLyS binding polypeptide from the BLyS and/or BLyS-like polypeptides, and

[0476] (d) recovering the dissociated, BLyS and/or BLyS-like polypeptide.

[0477] The invention also provides for kits containing a binding polypeptide of the invention for use in methods of detecting or isolating BLyS and/or BLyS-like polypeptides.

[0478] According to the invention, detection or isolation of BLyS target proteins comprises contacting a solution containing a BLyS target protein with a BLyS binding polypeptide. Depending on the particular application, the BLyS binding polypeptide may be free in solution or immobilized on a solid support or chromatographic material. Sufficient time is allowed to permit binding between the BLyS target protein and the binding polypeptides, and non-binding components in the solution or mixture are removed or washed away. The formation of a binding complex between the binding polypeptide and the BLyS target protein can then be detected, for example, by detecting the signal from a label on the binding polypeptide, which is one component of the binding complex. A label may be any label that generates a signal that can be detected by standard methods, such as a fluorescent label, a radioactive compound, or an enzyme that reacts with a substrate to generate a detectable signal. Suitable such labels are discussed above. A phage binding polypeptide according to the invention, that is, a recombinant phage displaying a BLyS binding polypeptide on its surface, may form a complex with BLyS and/or BLyS-like polypeptides that is detectable as a precipitate or sediment in a reaction tube, which can be detected visually after settling or centrifugation. Alternatively, a sandwich-type assay may be used, wherein a BLyS binding polypeptide is immobilized on a solid support such as a plastic tube or well, or a chromatographic support matrix such as agarose beads, then the solution suspected of containing the BLyS target is contacted with the immobilized binding polypeptide and non-binding materials or components are removed or washed away.

[0479] The binding polypeptides according to this invention are particularly useful for detection and/or isolation of BLyS and/or BLyS-like polypeptides by affinity chromatography methods. Any conventional method of chromatography may be employed. Preferably, a BLyS binding polypeptide of the invention will be immobilized on a solid support suitable, for example, for packing a chromatography column. The immobilized BLyS binding polypeptide affinity ligand can then be loaded or contacted with a feed stream under conditions favorable to formation of binding polypeptide/BLyS (or BLyS-like polypeptide) complexes. Non-binding materials can be washed away. Examples of suitable wash conditions can readily be determined by one of skill in the art and include but are not limited to [PBS/0.01% Tween 20, pH7.2] and [1M NaCl/10 mM Tris, pH7.5]. Tris wash buffers may be preferable since phosphates can preciptate in 50% ethylene glycol. In general, non-limiting terms, wash buffers are pH7.0, optionally containing 0.0 to 1.5 M NaCl, more preferably 1M NaCl. Additionally, wash buffers may optionally contain a mild detrgenet, such as, for example, Tween 20, Tween 80, or NP-80. BLyS or BLyS-like polypeptide can be eluted from the BLyS binding polypeptide by introducing solution conditions that favor dissociation of the binding complex. Suitable elution solutions can readily be determined by one of skill in the art and include but are not limited to [50% ethylme glycol/10 mM NaOAc]. By way of non-limiting example, useful elution buffers, for the purposes of the present invention contain 40-60% ethylene glycol, preferably 50% ethylene glycol.; and 50-100 mM NaOAc with a pH in the range of pH 4-pH7, more preferably, pH 4-pH 6 and most preferably pH 4.5-pH 5.5. Preferably, a fast flow affinity chromatographic technique is used to bind the molecules and from which purified BLyS or BLyS-like polypeptides are eluted.

[0480] Alternatively, batch chromatography can be carried out by mixing a solution containing the BLyS target and the BLyS binding polypeptide, then isolating complexes of the BLyS target and the binding polypeptides. For this type of separation, many methods are known. For example, the binding polypeptide may be immobilized on a solid support such as beads, then separated from the feed stream along with the BLyS target by filtration. In another example, the BLyS binding polypeptide may be modified with its own affinity tag, such as a polyHis tail or streptavidin binding region, which can be used to isolate the binding polypeptide after complexes have formed using an immobilized metal affinity chromatographic resin or steptavidin-coated substrate. Once separated, the BLyS target can be released from the binding polypeptide under elution conditions and recovered in a purified form.

[0481] Methods of producing BLyS or a BLyS-like polypeptides usually yield BLyS or BLyS-like polypeptides in a feed stream that additionally contains impurities (with respect to the BLyS target). One purpose of the present invention is to produce BLyS binding polypeptides and preparations (such as affinity chromatography media or surfaces) comprising BLyS binding polypeptides that allow rapid and highly specific purification of BLyS target proteins from a feed stream. BLyS binding polypeptides obtained herein may easily be tailored to isolate BLyS target protein from a particular feed stream, using or routinely modifying conditions and techniques known in the art. If an alternate production method for BLyS is used, producing a different feed stream, a different set of BLyS binding polypeptides and/or conditions may be necessary to achieve the same level of purification. The new set of BLyS binding polypeptides and/or conditions can be readily obtained following or modifying procedures outlined herein, or otherwise known in the art.

[0482] Kits

[0483] The present invention is also directed to an assay kit which can be useful in screening for the presence of BLyS and/or quantitating BLyS concentrations in a fluid, such as, for example, a biological fluid (e.g,. blood, serum, or synovial fluid).

[0484] In a particular embodiment of the present invention, an assay kit is contemplated which comprises in one or more containers of BLyS binding polypeptide(s) according to the invention and, optionally, a detection means for determining the presence of a BLyS target/BLyS binding polypeptide interaction or the absence thereof. The kit further optionally contains BLyS protein that may be used, for example as a control or standard. The BLyS binding polypeptide may be free or expressed on the surface of a host cell or on the surface of a bacteriophage.

[0485] In a specific embodiment, either the BLyS binding polypeptide or the BLyS protein is labeled. As further discussed herein, a wide range of labels can be used in accordance with the present invention, including but not limited to conjugating the recognition unit to biotin by conventional means. Alternatively, the label may comprise, e.g., a fluorogen, an enzyme, an epitope, a chromogen, or a radionuclide. Preferably, the biotin is conjugated by covalent attachment to either the BLyS binding polypeptide or the BLyS protein. Preferably, the BLyS binding polypeptide is immobilized on a solid support. The detection means employed to detect the label will depend on the nature of the label and can be any known in the art, e.g., film to detect a radionuclide; an enzyme substrate that gives rise to a detectable signal to detect the presence of an enzyme; antibody to detect the presence of an epitope, etc.

[0486] Methods of Screening for BLyS Binding Molecules

[0487] The present invention also encompasses screening methods for identifying polypeptides i and nonpolypeptides that bind BLyS, and the BLyS binding molecules identified thereby. This method comprises the steps of:

[0488] (a) contacting a BLyS protein or BLyS-like protein with a plurality of molecules; and

[0489] (b) identifying a molecule that binds the BLyS protein or BLyS-like protein.

[0490] The step of contacting the BLyS protein or BLyS-like protein with the plurality of molecules may be effected in a number of ways. For example, one may contemplate immobilizing the BLyS protein or BLyS-like protein on a solid support and bringing a solution of the plurality of molecules in contact with the immobilized BLyS protein or BLyS-like protein. Such a procedure would be akin to an affinity chromatographic process, with the affinity matrix being comprised of the immobilized BLyS protein or BLyS-like polypeptide. The molecules having a selective affinity for the BLyS protein or BLyS-like polypeptide can then be purified by affinity selection. The nature of the solid support, process for attachment of the BLyS protein or BLyS-like polypeptide to the solid support, solvent, and conditions of the affinity isolation or selection are largely conventional and well known to those of ordinary skill in the art.

[0491] Alternatively, one may also separate a plurality of polypeptides into substantially separate fractions comprising a subset of or individual polypeptides. For instance, one can separate the plurality of polypeptides by gel electrophoresis, column chromatography, or like method known to those of ordinary skill for the separation of polypeptides. The individual polypeptides can also be produced by a transformed host cell in such a way as to be expressed on or about its outer surface (e.g., a recombinant phage). Individual isolates can then be “probed” by the BLyS protein or BLyS-like polypeptide, optionally in the presence of an inducer should one be required for expression, to determine if any selective affinity interaction takes place between the BLyS protein or BLyS-like protein and the individual clone. Prior to contacting the BLyS protein or BLyS-like protein with each fraction comprising individual polypeptides, the polypeptides could first be transferred to a solid support for additional convenience. Such a solid support may simply be a piece of filter membrane, such as one made of nitrocellulose or nylon. In this manner, positive clones could be identified from a collection of transformed host cells of an expression library, which harbor a DNA construct encoding a polypeptide having a selective affinity for BLyS protein or BLyS-like protein. Furthermore, the amino acid sequence of the polypeptide having a selective affinity for the BLyS protein or BLyS-like protein can be determined directly by conventional means or the coding sequence of the DNA encoding the polypeptide can frequently be determined more conveniently. The primary sequence can then be deduced from the corresponding DNA sequence. If the amino acid sequence is to be determined from the polypeptide itself, one may use microsequencing techniques. The sequencing technique may include mass spectroscopy.

[0492] In certain situations, it may be desirable to wash away any unbound BLyS protein or BLyS-like protein, or alternatively, unbound polypeptides, from a mixture of the BLyS protein or BLyS-like protein and the plurality of polypeptides prior to attempting to determine or to detect the presence of a selective affinity interaction. Such a wash step may be particularly desirable when the BLyS protein or BLyS-like protein or the plurality of polypeptides is bound to a solid support.

[0493] The plurality of molecules provided according to this method may be provided by way of diversity libraries, such as random or combinatorial peptide or nonpeptide libraries which can be screened for molecules that specifically bind to BLyS. Many libraries are known in the art that can be used, e.g., chemically synthesized libraries, recombinant (e.g., phage display libraries), and in vitro translation-based libraries. Examples of chemically synthesized libraries are described in Fodor et al., Science, 251:767-773 (1991); Houghten et al., Nature, 354:84-86 (1991); Lam et al., Nature, 354:82-84 (1991); Medynski, Bio/Technology, 12:709-710 (1994); Gallop et al., J. Medicinal Chemistry, 37(9):1233-1251 (1994); Ohlmeyer et al., Proc. Natl. Acad. Sci. USA, 90:10922-10926 (1993); Erb et al., Proc. Natl. Acad. Sci. USA, 91:11422-11426 (1994); Houghten et al., Biotechniques, 13:412 (1992); Jayawickreme et al., Proc. Natl. Acad. Sci. USA, 91:1614-1618 (1994); Salmon et al., Proc. Natl. Acad. Sci. USA, 90:11708-11712 (1993); PCT Publication No. WO 93/20242; and Brenner and Lerner, Proc. Natl. Acad. Sci. USA, 89:5381-5383 (1992).

[0494] Examples of phage display libraries are described in Scott and Smith, Science, 249:386-390 (1990); Devlin et al., Science, 249:404-406 (1990); Christian et al., J. Mol. Biol., 227:711 -718 (1992); Lenstra, J. Immunol. Meth., 152:149-157 (1992); Kay et al., Gene, 128:59-65 (1993); and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.

[0495] In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991; and Mattheakis et al., Proc. Natl. Acad. Sci. USA, 91:9022-9026 (1994).

[0496] By way of examples of nonpeptide libraries, a benzodiazepine library (see, e.g., Bunin et al., Proc. Natl. Acad. Sci. USA, 91:4708-4712 (1994)) can be adapted for use. Peptoid libraries (see, Simon et al., Proc. Natl. Acad. Sci. USA, 89:9367-9371 (1992)) can also be used. Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (Proc. Natl. Acad. Sci. USA, 91:11138-11142 (1994)).

[0497] The variety of non-peptide libraries that are useful in the present invention is great. For example, Ecker and Crooke, Bio/Technology, 13:351-360 (1995) list benzodiazepines, hydantoins, piperazinediones, biphenyls, sugar analogs, beta-mercaptoketones, arylacetic acids, acylpiperidines, benzopyrans, cubanes, xanthines, aminimides, and oxazolones as among the chemical species that form the basis of various libraries.

[0498] Non-peptide libraries can be classified broadly into two types: decorated monomers and oligomers. Decorated monomer libraries employ a relatively simple scaffold structure upon which a variety functional groups is added. Often the scaffold will be a molecule with a known useful pharmacological activity. For example, the scaffold might be the benzodiazepine structure.

[0499] Non-peptide oligomer libraries utilize a large number of monomers that are assembled together in ways that create new molecular shapes that depend on the order of the monomers. Among the monomer units that have been used are carbamates, pyrrolinones, and morpholinos. Peptoids, peptide-like oligomers in which the side chain is attached to the alpha amino group rather than the alpha carbon, form the basis of another version of non-peptide oligomer libraries. The first non-peptide oligomer libraries utilized a single type of monomer and thus contained a repeating backbone. Recent libraries have utilized more than one monomer, giving the libraries added flexibility.

[0500] Screening the libraries can be accomplished by any of a variety of commonly known methods. See, e.g., the following references, which disclose screening of peptide libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol., 251:215-218; Scott and Smith, 1990, Science, 249:386-390; Fowlkes et al., 1992; BioTechniques, 13:422-427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA, 89:5393-5397; Yu et al., 1994, Cell, 76:933-945; Staudt et al., 1988, Science, 241:577-580; Bock et al., 1992, Nature, 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA, 89:6988-6992; Ellington et al., 1992, Nature, 355:850-852; U.S. Pat. No. 5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all to Ladner et al.; Rebar and Pabo, 1993, Science, 263:671-673; and CT Publication No. WO 94/18318.

[0501] In a specific embodiment, screening to identify a molecule that binds BLyS can be carried out by contacting the library members with a BLyS protein or BLyS-like protein immobilized on a solid phase and harvesting those library members that bind to the BLyS protein or BLyS-like protein. Examples of such screening methods, termed “panning” techniques are described by way of example in Parmley and Smith, 1988, Gene, 73:305-318; Fowlkes et al., 1992, BioTechniques, 13:422-427; PCT Publication No. WO 94/18318; and in references cited herein.

[0502] In another embodiment, the two-hybrid system for selecting interacting proteins in yeast (Fields and Song, 1989, Nature, 340:245-246; Chien et al., 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) can be used to identify molecules that specifically bind to BLyS or BLyS-like proteins.

[0503] An alternative screening method for obtaining new binding moieties capable of binding to BLyS target proteins is to employ a competition assay, in which a BLyS target is bound to a BLyS binding polypeptide according to the present invention, preferably labeled, and then the complex is exposed to one or more test moieties. Succesful new BLyS binding moieties will be test moieties capable of effectively competing for binding to the BLyS target in the presence of a known BLyS binder disclosed herein.

[0504] Polypeptides specifically binding BLyS target proteins can be conveniently selected from any peptide library, including random peptide libraries, combinatorial peptide libraries, or biased peptide libraries. The term “biased” is used herein to mean that the method of generating the library is manipulated so as to restrict one or more parameters that govern the diversity of the resulting collection of molecules, in this case peptides.

[0505] Thus, a truly random peptide library would generate a collection of peptides in which the probability of finding a particular amino acid at a given position of the peptide is the same for all 20 amino acids. A bias can be introduced into the library, however, by specifying, for example, that a lysine occur every fifth amino acid or that positions 4, 8, and 9 of a decapeptide library be fixed to include only arginine. For libraries designed to display a stable loop structure, a peptide sequence may be designed to include two invariant cysteine residues, with all other amino acid positions permitting one or more amino acid residues but excluding cysteine residues. (See, Example 1, infra.) Clearly, many types of biases can be contemplated, and the present invention is not restricted to any particular bias. Furthermore, the present invention contemplates specific types of peptide libraries, such as phage displayed peptide libraries and those that utilize a DNA construct comprising a lambda phage vector with a DNA insert.

[0506] As mentioned above, in the case of a BLyS binding molecule that is a polypeptide, the polypeptide may have about 6 to less than about 60 amino acid residues, preferably about 6 to about 10 amino acid residues, and most preferably, about 6 to about 22 amino acids. In another embodiment, a BLyS binding polypeptide has in the range of 15-100 amino acids, or 20-50 amino acids.

[0507] The selected BLyS binding polypeptide can be produced by chemical synthesis or recombinant expression, as described above.

[0508] The specific BLyS binding polypeptides disclosed herein were isolated using phage display technology, to identify BLyS binding polypeptides exhibiting particular preselected binding properties. These BLyS binding polypeptides were isolated initially by screening nine phage display libraries, that is, populations of recombinant bacteriophage transformed to express an exogenous recombinant polypeptide on their surface. In order to isolate new polypeptide binding moieties for a particular target, such as BLyS, screening of peptide libraries, for example using phage display techniques, is especially advantageous, in that very large numbers (e.g., 5×10⁹) of potential binders can be tested and successful binders isolated in a short period of time.

[0509] In order to prepare a phage library of potential binding polypeptides to screen for members of the library that are BLyS binding polypeptides, a candidate binding domain is selected to serve as a structural template for the polypeptides to be displayed in the library. The phage library is made up of polypeptide analogues of this template or “parental binding domain.”The parental binding domain is a polypeptide molecule that may be a naturally occurring or synthetic protein or polypeptide, or polypeptide region or domain of a protein. The parental binding domain may be selected based on knowledge of a known interaction between the parental binding domain and a target protein, but this is not critical. In fact, it is not essential that the parental binding domain have any affinity for a target at all because its purpose is to provide a structure from which a multiplicity of polypeptide analogues (a “library”) can be generated, which multiplicity of polypeptide analogues will include one or more binding polypeptides that exhibit the desired binding and release properties with respect to BLyS target proteins (and any other properties selected).

[0510] Knowledge of the exact polypeptide that will serve as the parental binding domain, or knowledge of a class of proteins or domains to which the parental binding domain belongs can be useful in determining the conditions under which BLyS binding polypeptides optimally bind BLyS target proteins as well as the conditions under which BLyS binding polypeptides optimally release BLyS target proteins. Similarly, the binding and/or release conditions may be selected with regard to known interactions between a binding domain and the BLyS target protein, for example, to favor the interaction under the binding and/or release conditions, or they may be selected without regard to such known interactions. Likewise, the parental binding domain can be selected taking into account a desired binding and/or release condition or not. It is understood that if the binding domain analogues of a library are unstable under a proposed or desired binding or release condition, no useful binding polypeptides may be obtained.

[0511] In selecting the parental binding domain, the most important consideration is how the analogue domains will be presented to the BLyS target protein, that is, in what conformations the BLyS target and the polypeptide analogues will contact one another. In preferred embodiments, for example, the polypeptide analogues will be generated by insertion of synthetic DNA encoding the polypeptide analogue into a replicable genetic package, resulting in display of the domain on the surface of a microorganism, such as M13 phage, using techniques as described in Kay et al., Phage Display of Peptides and Proteins: A Laboratory Manual (Academic Press, Inc.; San Diego 1996) and U.S. Pat. No. 5,223,409 (Ladner et al.), incorporated herein by reference. For formation of phage display libraries, it is preferred to use structured polypeptides as the parental binding domain or template, as opposed to unstructured, linear peptides. Mutation of surface residues in a protein domain or polypeptide molecule will usually have little effect on the overall structure or general properties (such as size, stability, and temperature of denaturation) of the protein; while at the same time mutation of surface residues may profoundly affect the binding properties of the molecule. The more tightly a polypeptide segment is constrained, the less likely it is to bind to any particular target. If it does bind, however, the binding is likely to be tighter and more specific. Thus, it is preferred to select a parental binding domain wherein the parental polypetide has structure and, thereby in turn, select a structure for the polypeptide analogues of the library, which is constrained within a framework having some degree of rigidity.

[0512] Preferably the protein domain that is used as the template or parental domain for generating the library of domain analogues will be a peptide molecule that is a relatively small protein or polypeptide. Small polypeptides offer several advantages over large proteins: First, the mass per binding site is reduced. Highly stable protein domains having low molecular weights, for example, Kunitz domains (˜7 kilodaltons, kDa), Kazal domains (˜7 kDa), Cucurbida maxima trypsin inhibitor (CMTI) domains (˜3.5 kDa), and endothelin (˜2 kDa), can show much higher binding per gram than do antibodies (150 kDa) or single chain scFv antibodies (30 kDa). Second, the possibility of non-specific binding is reduced because there is less molecular surface available for nonspecific binding. Third, small polypeptides can be engineered to have unique tethering sites in a way that is impracticable for larger proteins or antibodies. For example, small proteins and polypeptides can be engineered to have lysines only at sites suitable for tethering to a chromatography matrix. This is not feasible for antibodies. Fourth, a constrained polypeptide structure is more likely to retain its functionality when transferred (with the structural domain intact) from one framework to another. For instance, the binding domain structure is likely to be transferable from the framework used for presentation in a library, such as displayed on a phage, to an isolated protein removed from the presentation framework or immobilized on a chromatographic substrate.

[0513] In specific embodiments, the BLyS binding polypeptides of the invention are immobilized. BLyS binding polypeptide molecules according to the invention may be immobilized, for example, on chromatographic support materials to form efficient BLyS separation or affinity chromatographic media. Immobilized BLyS binding polypeptides of the invention have uses that include, but are not limted to, detecting, isolating or removing BLyS target proteins from solutions. One strategy for generating BLyS binding polypeptide molecules that can be immobilized, for example, on matrices, resins, or supports, involves selecting appropriate binding domain templates such that BLyS binding polypeptide molecules are generated that have one or more amino acids that may be used to covalently link the BLyS binding polypeptide to a chromatographic resin or substrate to form an affinity resin. Similarly, the N-terminal amino group or the C-terminal carboxyl group of a peptide molecule may be modified by adding a capping group to render it inert or a functional group, which permits linkage to a support medium. For example, the C-terminal carboxyl group of a protein domain may be converted to an amide or a hydrazide (—NH—NH₂) group for reaction with an aldehyde-functional substrate or other amine-reactive substrate. This technique is preferred. Another preferred modification of BLyS binding polypeptides useful for linking a BLyS binding polypeptide molecule of the invention to a chromatography material is a polypeptide linker comprising, or alternatively consisting of, the amino acid sequence Pro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ ID NO: 13).

[0514] In one non-limiting example of a screening procedure to obtain BLyS binding polypeptides encompassed by the invention, the phage in a phage display library are contacted with and allowed to bind a BLyS target protein that is immobilized on a solid support. Those phage that display non-binding polypeptides are separated from those that bind the BLyS target protein. Any of various techniques known in the art may be applied to dissociate the bound phage from the immobilized BLyS protein, and to collect and/or amplify the phage and/or their nucleic acid contents. Using these techniques it is possible to identify a BLyS binding phage that is about 1 in 20 million in the population. Libraries, displaying 10-20 million or more potential binding peptide molecules each, are rapidly screened to find high-affinity BLyS binding polypeptides.

[0515] In each round of screening, the diversity of a population falls until only efficient binding polypeptides remain, that is, the process converges. Typically, a phage display library will contain several closely related binding polypeptides (10 to 50 different binding polypeptides out of 10 million). Indications of convergence include increased binding (measured by phage titers) and recovery of closely related sequences. After a first set of binding polypeptide molecules is identified, the sequence information can be used to design other libraries biased for members having additional desired properties, for example, discrimination between different forms of BLyS (e.g., the membrane form and the soluble form of BLyS) and fragments thereof, or discrimination between BLyS and closely related impurities in a feed stream.

[0516] Such techniques make it possible not only to screen a large number of potential binding polypeptides, but make it practical to repeat the binding and elution cycles and to build secondary, biased libraries for screening polypeptide analogue-displaying phage that meet specific criteria. Using these techniques, a polypeptide analogue biased library may be screened to reveal members that bind tightly, that is, have high affinity for BLyS target protein, under the screening conditions.

[0517] In the present invention target BLyS protein molecules were biotinylated and then bound to streptavidin-coated magnetic particles. Nine phage display libraries of different design were screened for the ability to bind the immobilized BLyS. Each library was characterized by M13 phage displaying variegated peptides of different lengths and overall structure: A library designated TN6/6 (2×10⁸ variants) displayed a variegated 12-mer with two internal invariant cyteines to form a hexamer loop structure. A library designated TN7/4 (2.3×10⁹ variants) presented a variegated 13-mer having two internal invariant cyteines to form a heptamer loop structure. A library designated TN8/9 (5×10⁹ variants) displayed a variegated 14-mer with two internal invariant cyteines to form an octamer loop structure. A library designated TN9/4 (3.2×10⁹ variants) presented a variegated 1 6-mer having two internal invariant cyteines to form a nonamer loop structure. A library designated TN10/9 (2.5×10⁹ variants) displayed a variegated 16-mer with two internal invariant cyteines to form a decamer loop structure. A library designated TN12/1 (1.4×10⁹ variants) presented a variegated 18-mer having two internal invariant cyteines to form a dodecamer loop structure. A library designated as Substrate Phage Library #2, having a diversity of about 2×10⁸ amino acid sequences, was designed to include a linear peptide-variegated region in the display polypeptide consisting of 13 consecutive amino acids, and the display polypeptide design allowed any amino acid residue except cysteine to occur at each position. Finally, two commercially available linear phage display libraries were also screened, designated PhD 7 and PhD 12, respectively (New England Biolabs). The PhD 7 library displayed a linear random-sequence 7-mer; the PhD 12 libary displayed a random-sequence 12-mer.

[0518] BLyS binding phage were isolated and collected from all of the libraries except PhD 7.

[0519] After analysis of the sequences isolated from the library screenings, several families of BLyS binding peptides were defined (see, consensus sequences A-G and H-L, above). The amino acid sequences of the BLyS-binding “hits” from the first rounds of screening are set forth in Tables 1-8 (infra).

[0520] In order to obtain BLyS binding polypeptides having an even higher affinity for BLyS targets, a specialized library was prepared, i.e., a BLyS affinity maturation library, based on variegation of high affinity examplars of the PhD 12 library (see Example 6). This library was designed to provide a population enriched with polypeptides likely to show high affinity for BLyS. The selections from this library were performed to eliminate, by prolonged competition with soluble eluants of soluble BLyS or other BLyS binding polypeptides, all but the polypeptides having the highest affinity for BLyS. A large family of high affinity BLyS binding polypeptides was isolated from four rounds of screening the affinity maturation library, and their amino acid sequences appear in Table 14 (infra).

[0521] BLyS binding polypeptides according to the invention and phage comprising such polypeptides have uses that include, but are not limited to, detecting and isolating BLYS and BLyS-like polypeptides, as described above.

[0522] Isolation of BLyS binding polypeptides and their use in accordance with this invention will be further illustrated below. The specific parameters included in the following examples are intended to illustrate the practice of the invention, and they are not presented to in any way limit the scope of the invention.

EXAMPLE 1

[0523] Screening of Phage Display Libraries

[0524] Streptavidin-coated magnetic beads (Dynal M-280) were chosen for presentation of the target during screening because of their superior binding capacity compared to that of a 96 well plate. The binding capacity of the beads for biotinylated antibodies was 5-10 μg/mg of beads as stated by the manufacturer. For this study and the screening to follow, 5 μg of biotinylated recombinant BLyS (obtained from Human Genome Sciences, Inc.) was allowed for each mg of beads. This amount of biotinylated BLyS represents a 10-fold excess of target, for saturation of the beads. Unbound BLyS was washed away. Bound biotinylated BLyS was confirmed with detection using Mab 16C9 (murine anti-BLyS, Human Genome Sciences) primary antibody and a goat anti-mouse HRP conjugate as the secondary antibody. An irrelevant monoclonal antibody (anti-TNFα) was used to probe a second set of beads to control for nonspecific binding. The color reagent TMB was used and the assay read at OD 630 nm.

[0525] Nine libraries, TN6/6, TN7/4, TN8/9, TN9/4, TN10/9, TN12/1, Substrate Phage #2, PhD7, and PhD12, were screened for BLyS binders. The makeup of these libraries was as follows:

[0526] The TN6/6 phage display library was composed of recombinant M13 phage displaying variegated peptides with the potential to form loop structures based on a polypeptide template having the structure Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa (SEQ ID NO: 14) and providing 2.0×10⁸ peptide diversity.

[0527] The TN7/4 phage display library was composed of recombinant M13 phage displaying variegated peptides with the potential to form loop structures based on a polypeptide template having the structure Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa (SEQ ID NO: 15) and providing 2.3×10⁹ peptide diversity.

[0528] The TN8/9 phage display library was composed of recombinant M13 phage displaying variegated peptides with the potential to form loop structures based on a polypeptide template having the structure Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa (SEQ ID NO: 16) and providing about 5×10⁹ peptide diversity.

[0529] The TN9/4 phage display library was composed of recombinant M13 phage displaying variegated peptides with the potential to form loop structures based on a polypeptide template having the structure Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa (SEQ ID NO: 17) and providing about 3.2×10⁹ peptide diversity.

[0530] The TN10/9 phage display library was composed of recombinant M13 phage displaying variegated peptides with the potential to form loop structures based on a polypeptide template having the structure Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa (SEQ ID NO: 18) and providing 2.5×10⁹ peptide diversity.

[0531] The TN12/1 phage display library was composed of recombinant M13 phage displaying variegated peptides with the potential to form loop structures based on a polypeptide template having the structure Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa (SEQ ID NO: 19) and providing 1.4×10⁹ peptide diversity.

[0532] Substrate Phage Library #2 was composed of recombinant M13 phage displaying a polypeptide insert of approximately 80 amino acids, having two streptavidin binding domains, a linear variegated segment of thirteen amino acids where all amino acids except Cys were permitted at each position, and a Factor Xa cleavage site, linked together with peptide linkers. This library provided a diversity of 2>10⁸ display polypeptides.

[0533] Libraries PhD7 and PhD12 were composed of recombinant M13 phage displaying randomized linear seven- and twelve-amino acid peptides, obtained commercially from New England Biolabs.

[0534] Prior to each round of screening, phage libraries or phage library pools were depleted of phage capable of binding the streptavidin beads by sequentially adding the libraries to 5 separate aliquots of streptavidin beads and allowing them to bind for 10 minutes. The depleted libraries were added to biotinylated BLyS on streptavidin magnetic beads and allowed to bind for 1 hour at room temperature. For round 1 of the screening, all the libraries were kept separate except PhD7 and PhD12 which were pooled from the beginning. After binding, the beads were washed 7 times and bound phage were incubated for 10 minutes with citrate buffered saline at pH 2.0 to elute. The eluted phage were neutralized with 2 M Tris-HCl pH 8.0 and allowed to infect E. coli XL-1 Blue MRF′. The infected cells were spread on a large agar plate and standard phage techniques known in the art were used to produce the starting material for the next round. For each round of screening the fraction of input recovered was calculated for each library (library pool). This is equal to the number of phage recovered divided by the number on starting phage. No further rounds of screening were done after the fraction of input recovered reached 1×10⁻² total phage. Pool A for round 2 of screening was a mixture of TN6/6, TN7/4 and TN8/9 round 1 outputs. Pool B for round 2 was a mixture of TN9/4, TN10/9, and TN12/1 round 1 outputs. After round 2 on Pool A and Pool B the fraction of input recovered was equal to or greater than 1×10⁻² and no further rounds were done. For the Substrate Phage Library #2 and the PhD pool a third round of screening was required.

[0535] At the conclusion of screening individual phage isolates were randomly selected and tested by ELISA for binding to BLyS. The same isolates were submitted for DNA sequence analysis to identify the nucleotide and deduced amino acid sequence of the displayed peptide. Isolates were also tested for their ability to bind to recombinant BLyS in feed streams of CHO supernatant and Sf9 supernatant (supplied by Human Genome Sciences, Inc.).

[0536] Each isolate was tested for binding to BLyS by standard ELISA techniques where bound phage were detected with a monoclonal anti-phage antibody/HRP conjugate. Approximately 90% of the isolates from the TN libraries Pool A and Pool B had binding signals on BLyS ranging from 3× to 12× above the background binding on streptavidin alone. Isolates from the Substrate Phage Library showed similar but slightly lower binding signals.

[0537] To assess the ability of the BLyS binding polypeptides to recognize the BLyS target in potential process feed streams, phage binding was determined in two feed streams: CHO and Sf9 supernatants spiked with BLyS protein. Phage were allowed to bind to BLyS in either CHO supernatant or Sf9 supernatant rather than the standard conditions of PBS plus Tween. All other wash steps were the same as the standard ELISA conditions. The binding of BLyS binding polypeptides to BLyS in PBS+Tween, CHO supernatant, and Sf9 supernatant binding was very similar under all conditions. Several BLyS binding polypeptide isolates demonstrated reduced binding of up to 50% in the CHO supernatant. Isolates of BLyS binding polypeptides binding to BLyS in the Sf9 supernatant was not significantly different from binding under the standard conditions. The PhD and Substrate Phage Library isolates were also compared in the feed streams. Remarkably, several of these isolates exhibited greater binding in the feed streams than under the standard conditions.

[0538] Amino acid sequences of the displayed peptides were derived from sequencing the phage isolate DNA inserts. Sequence data from the phage isolates were grouped by library and sorted according to the degree of similarity. The BLyS binding phage isolate peptides are shown in Tables 1-8 below. These peptides represent the translation of the DNA sequences across the varied regions of the genes encoding the phage display fusion/peptide. TABLE 1 TN6/6 Library BLyS-binding Sequences Phage Isolate Amino Acid Sequence SEQ ID NO: 453-01-B06 HLRCWSTNCRYD 20 453-01-A04 VMDCLINRCDTV 21

[0539] TABLE 2 TN7/4 Library BLyS-binding Sequences Phage Isolate Amino Acid Sequence SEQ ID NO: 453-01-B04 KSKCFFPWECQQA 22 453-01-D11 AMKCYFPWECANG 23 453-01-A05 NVACYFPWECHHP 24 453-01-D01 NAPCYFPWECFSI 25 453-01-D03 SVNCWFPWECVGN 26 453-01-A08 KEPCYFYWECAVS 27

[0540] TABLE 3 TN8/9 Library BLyS-binding Sequences Phage Isolate Amino Acid Sequence SEQ ID NO: 453-01-D04 DTNCDLLTKMCGPQ 28 453-01-C06 GTPCDLLTKLCLLW 29 453-01-D10 MSECDLLTKICLMG 30 453-01-B07 VPFCDLLTKHCFEA 31 453-01-B09 VPFCDLLTKHCFEA 32 453-01-C02 WSACDLLTKQCVQV 33 453-01-A06 -DGCDELTKICGMK 34 453-01-B03 KSWCDELTKVCFDP 35 453-01-B11 KWMCDELTKQCQYV 36 453-01-A02 MKYCDELTKICVGW 37 453-01-B05 YFQCDELTKMCWQK 38 453-01-A11 AMHCDKLTKHCKFH 39 453-01-A03 VPYCDKLTKICQW- 40 453-01-A07 EVFCDVLTKVCFHD 41 453-01-C09 KPKCDVLTKMCDWL 42 453-01-B02 TQHCDVLTKQCFTI 43 453-01-C01 GHFCDRLTKYCFEP 44 453-01-A09 HIQCDRLTKSCLSV 45 453-01-D05 IKACDILTKVCWPP 46 453-01-A01 QFDCDPLTKYCGEF 47 453-01-C07 KMYCDHLTGYCWPE 48 453-01-C11 MQSCDILTGYCFKR 49 453-01-D12 GPWCDILTGFCLAQ 50 453-01-C04 SVRCDLLTGWCPVW 51 453-01-B10 PADCDPLTNICFWK 52 453-01-D02 TNVCDPLTNVCFMN 53 453-01-C05 EHWCDDLTHLCFRL 54 453-01-D08 GYWCDVLTNNCWKI 55 453-01-C10 LYNCDYLTRLCFEP 56 453-01-C08 HVDCLLHPKACYKY 57 453-01-D07 VQDCLLHPKACQMQ 58 453-01-D09 KFDCLLKPMFCSNH 59 453-01-C12 FADCLIHPKSCKPL 60 453-01-D06 HGNCYPFPWECESK 61 453-01-B01 MIIVLLLLRFAISR 62 453-01-A12 SLLVIFLLIGAGSL 63

[0541] TABLE 4 TN9/4 Library BLyS-binding Sequences Phage Isolate Amino Acid Sequence SEQ ID NO: 453-01-G06 FHPCDMLTGIWCQPN 64 453-01-H01 SKRCDLLTKMWCETE 65 453-01-F02 TKFCDRLTMPKCVWK 66 453-01-E03 NTFCPDPLTGRCVNP 67 453-01-E11 DWTCDPLFHRECIFE 68 453-01-H09 PQPCDLLFEKKCSIK 69 453-01-H02 RWHCDMLINPSCLPD 70 453-01-E04 KIQCDIVNLSSCVYP 71 453-01-G11 LNACDIVHPNYCSGM 72 453-01-F01 AKACSIVNLESCEYL 73 453-01-H06 RQACSIITPWGCPIP 74 453-01-F10 ADNCTVATLDFCYWT 75 453-01-E05 KPECNITKPQFCFGE 76

[0542] TABLE 5 TN10 Library BLyS-binding Sequences Phage Isolate Amino Acid Sequence SEQ ID NO: 453-01-H07 -NNCQWDELTSMCDPF 77 453-01-F05 SRLCHMDELTHVCVHF 78 453-01-F09 SRPCQIDELTKACFYN 79 453-01-G09 DRVCKLDFLTYNCLNH 80 453-01-F04 HSNCIMDLLTNRCFYD 81 453-01-H03 PFNCFHDPLTGLCLHS 82 453-01-F03 YDSCTYDRLTKQCYPS 83 453-01-F07 FHDCMYDALLGYCLPY 84 453-01-G08 NRSCDPLTRPKSCGL 85 453-01-G04 LSNCDWDDLIRQCLHD 86 453-01-E01 FWDCLFHPNSRYCVLS 87 453-01-E10 SRDCLLSPAMAWCGLD 88

[0543] TABLE 6 TN12/1 Library BLyS-binding Sequences Phage Isolate Amino Acid Sequence SEQ ID NO: 453-01-H05 GGNCYTDSLTKLHFCMGD 89 453-01-H04 --MCPRDPLTKAKLCNWH 90 453-01-G03 PNQCQDDLTKQWYSCHYH 91 453-01-F11 FDMCFDALTKQNFYCRFH 92 453-01-F06 RNMCVDRLTKLQHGCEGA 93 453-01-G07 DPECLTSFDRLTKMCWPW 94 453-01-H11 DDECHYDYLTHYMRCDYR 95 453-01-G05 FGGCNIDLLTNTMMCHRN 96 453-01-G10 HGPCYWDELTMQWHCNHH 97 453-01-H12 GAMCVDLLTYTFRPCMYA 98 453-01-E07 SNKCWDELTHAWAECGRF 99 453-01-E09 RPVCYKGYDILTTQCMPW 100 453-01-G01 PSRCWFDLLFNKFVCKRN 101 453-01-H08 RSGCVYDMLLMTMYCPSN 102 453-01-H10 SNRCEGDQLMRPPSCRHL 103 453-01-F08 YRMCWWDDLLRGFVCDFH 104 453-01-E06 HDGCYDELLYRWTRCEHR 105 453-01-E08 WAWCFDELVQRYFTCFDH 106 453-01-E02 LPECRQYFPWEKQVCSYW 107

[0544] TABLE 7 PhD 12 Library BLyS-binding Sequences Phage Isolate Amino Acid Sequence SEQ ID NO: 453-02-B05 VHYDSLTKMWTR 108 453-02-D09 FTDPLTKMSLHS 109 453-02-C12 GYDVLTKLYFVP 110 453-02-A05 YYDRLTKLYSSM 111 453-02-B06 L?KDPLTKLYIS 112 453-02-A04 GYDVLTKL?FVP 113 453-02-B03 RLYDPLTKLVLS 114 453-02-B01 MFDPLTKIAFPA 115 453-02-D04 FYDSLTKTNLRD 116 453-02-B02 GIYDKLTRAWLP 117 4S3-02-B08 KYDPLTRAR?PL 118 453-02-D06 YIDQLTRLSLPS 119 453-02-A09 HqTFDILTRLHF 120 453-02-B04 WQFDVLTRSWTP 121 453-02-A02 GAAYDHLTRTWL 122 453-02-D05 YFDQLTHLSIKK 123 453-02-A06 AWDPLTMLVLPW 124 453-02-D03 ALWMDPLTGLAF 125 453-02-B12 WQFDVLT?SWTP 126 453-02-A01 WTDPLTHMEIYH 127 453-02-C04 WTDSLTGLWFPD 128 453-02-C05 YTDPLTGIV?PF 129 453-02-D08 YWDKLTMLHLGV 130 453-02-D02 YYDFLTRTVLPS 131 453-02-A03 RLDPLSKNDFPR 132 453-02-A11 LRYDPLLKS?IY 133 453-02-D07 LRYDPLLKSYIY 134 453-02-A07 YFDQFTHLSIKK 135 453-02-C08 YFDQ?THLSIKK 136

[0545] TABLE 8 Substrate Phage Library BLyS-binding Sequences Phage Isolate Amino Acid Sequence SEQ ID NO: 453-02-E04 EHYYTDPLTGARI 137 453-02-F01 EHY?TDPLTGARI 138 453-02-E09 EHYSTDPLTGARI 139 453-02-E07 EHYYTDPL?G?RI 140 453-02-G05 EHYYTDPL?G?R? 141 453-02-G09 EHYYTDPL?GAR? 142 453-02-E06 EH?YTDPLNGAR? 143 453-02-E05 EHYYNDPLNGAR? 144 453-02-F04 ?H?YNDPLNGAR? 145 453-02-G07 KPYYDPITKMTHH 146 453-02-F06 KPYYDPITKMSHH 147 453-02-E08 KPYYDPISKMTHH 148 453-02-G08 KP??DPISKMTHH 149 453-02-E01 QIGYDELTKAWVT 150 453-02-G02 QLGYDELTKAWVT 151 453-02-H06 KIDEL?MQNIIIW 152 453-02-F08 DHTDPLIQGLTKR 153 453-02-H01 WHDPLKHMHFHHE 154 453-02-F03 KHIDMETGLILQN 155 453-02-G03 MQVDPETGLKYEH 156 453-02-E03 ?LDQHVN???YQS 157 453-02-F10 E???T??LTGAR? 158 453-02-F02 GPYNI?RL?GEr? 159 453-02-E02 HIKMLHQGSFVGV 160 453-02-H08 HPTNT??HQ?VYS 161 453-02-H05 HRGQV??LNGMv? 162

[0546] A small number of phage isolates were selected for further characterization based upon several criteria: the strength of the ELISA signal (i.e., OD₆₃₀≧0.8 after 10 min.), the number of times the identical sequence was found, and the presence of a recurrent sequence motif. Some characteristics of the phage isolates that were selected are shown below. Sequence motifs found multiple times in the isolates are underlined. SEQ ELISA ID Isolate # signal Sequence NO: TN7-01-A08 1 1.2 KEPCYFYWECAVS  27 TN7-01-D11 2 1.0 AMKCYFPWECANG  23 TN7-01-B04 2 0.8 KSKCFFPWECQQA  22 TN8-01-C08 2 1.2 HVDCLLHPKACYKY  57 TN8-01-B07 2 1.4 VPFCDLLTKHCFEA  31 TN9-01-G06 1 1.2 FHPCDMLTGIWCQPN  64 TN9-01-011 1 0.8 LNACDIVHPNYCSGM  72 TN10-01-F05 1 1.0 SRLCHMDELTHVCVHF  78 TN12-01-H05 1 1.0 GGNCYTDSLTKLHFCMG  89 D PhD-02-B02 4 0.6 GIYDKLTRAWLP 117 PhD-02-B05 9 0.6 VHYDSLTKMWTR 108 PhD-02-C12 3 0.8 GYDVLTKLYFVP 110 PhD-02-D05 3 0.8 YFDQLTHLSIKK 123 PhD-02-C04 1 1.0 WTDSLTGLWFPD 128

[0547] Various release conditions (see below) were tested, in order to discover possible elution conditions where the BLyS binding polypeptides could be used as affinity ligands for BLyS purification. For release studies, a constant number of phage were applied to wells containing biotinylated BLyS immobilized on streptavidin. After allowing the phage to bind, each phage isolate was then “eluted” from the well with two five-minute washes using various buffers. Wells were washed with standard wash buffer, and bound phage were detected with a standard phage ELISA. Elution conditions were selected based on low pH release, which was the mechanism employed during screening, and alternative elution conditions based on conditions where the BLyS product was known to be stable for at least several hours. The various elution conditions were: PBS pH 7.0, citrate buffered saline pH 5.0, citrate buffered saline pH 3.0, citrate buffered saline pH 2.0, 1 M Guanidine pH 7.0, and 1 M urea pH 7.0. Binding of several of the isolates was reduced under the standard conditions (PBS pH 7.0). This may have occurred because these experiments were performed with concentrated phage preparations rather than the overnight bacterial supernatants used for all previous experiments. It is believed that the polyethylene glycol used to concentrate the phage interfered with the binding of these isolates. For the purposes of these release studies, the phage isolates selected had a starting signal of 0.5 OD 630 nm or greater. Overall, 1 M urea, pH 7.0 appeared to be the best buffer to release bound phage.

EXAMPLE 2

[0548] Immobilization of BLyS Binding Polypeptides on Sepharose-4FF Beads

[0549] On the basis of the above results, six display phage sequences were chosen for further study:

[0550] TN7-01-A08 (SEQ ID NO: 27), TN8-01-B07 (SEQ ID NO: 31), TN10-01-F05 (SEQ ID NO: 78), TN12-01-H05 (SEQ ID NO: 89), PhD-02-C04 (SEQ ID NO: 128), and PhD-02-C12 (SEQ ID NO: 110). In order to develop a suitable BLyS affinity ligand, the identified display peptides were synthesized to order by a commercial vendor, with slight modifications:

[0551] Two amino acids of leader were added to each binding peptide at the N-terminus, in order to avoid leaving a free amine at the first amino acid of the sequence corresponding to the variegated region of the phage display template; the N-terminus was acetylated to prevent immobilization of the peptide to the chromatographic matrix through that position; a C-terminal linker was added (i.e., -PGPEGGGK; SEQ ID NO: 13); and any internal lysines in the peptide were blocked with the group: ivDde (i.e., 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methyl butyl-L-lysine). This group was intact on the finished synthesized peptides and was removed after immobilization or fluorescein labeling. As an alternative modification, peptides with internal lysines were also synthesized with C-terminal hydrazide functional groups, which could be immobilized onto activated aldehyde chromatographic media.

[0552] The peptides were immobilized onto NHS-activated SEPHAROSE-4 Fast Flow agarose media (Pharmaceia) at ligand densities targeted to 2 μmol/ml. Actual ligand densities of peptides on the media ranged from 0.76,μmol/ml to 1.98 μmol/ml, as determined by amino acid analysis of immobilized peptide. All but one peptide was immobilized in aqueous conditions of 100 mM KH₂PO₄/150 mM NaCl/0.05% Tween 20, pH 7.5. For solubility reasons, the peptide DX217 (see, Table 9, below) was immobilized in 30% dimethyl formamide(DMF)/100 mM KH₂PO₄/150 mM NaCl/0.05% Tween 20. pH 7.5. Immobilization reactions were allowed to proceed for 2 hours at ambient temperature, followed by brief washing with pH 7.5 buffer. The Fast Flow SEPHAROSE media was then allowed to tumble at ambient temperature overnight to hydrolyze remaining NHS esters after which the media was washed to remove any unbound peptide. A solution of 2% hydrazine/DMF was used to de-block ligands containing ivDde-lysine. Media was then further washed with aqueous buffers and stored at 4° C. until packed into columns. Table 9 shows the sequences of the synthesized peptides and their measured densities on the agarose media. TABLE 9 BLyS Binding Peptides Synthesizes as Affinity Ligands Pep- SEQ tide Isolate Sequence ID Name source (potential disulfide loop underlined) NO: DX212 01-A08 Ac-AGKEPCYFYWECAVSGPGPEGGGK 163 DX214 01-B07 Ac-AGVPFCDLLTKHCFEAGPGPEGGGK 164 DX216 01-F-5 Ac-GSSRLCHMDELTHVCVHFAPPGPEGGGK 165 DX217 01-H05 Ac-GDGGNCYTDSLTKLHFCMGDEPGPEGGGK 166 DX219 02-C12 Ac-GYDVLTKLYFVPGGPGPEGGGK 167 DX221 02-C04 Ac-WTDSLTGLWFPDGGPGPEGGGK 168

[0553] BLyS-Ligand Affinity Determination (Overview of Procedure)

[0554] Dissociation constants between the synthetic peptides and BLyS (free in solution) were measured by fluorescence anisotropy (FA). In these experiments, the concentration of the fluorescein-labeled peptide is held constant and the BLyS protein concentration was varied. The observed change in anisotropy is fit to the following equation via nonlinear regression to obtain the apparent K_(D). $\begin{matrix} {{Peptide} + {{{BLyS}\underset{K_{D}}{}{Peptide}} \cdot {BLyS}}} \\ {r_{obs} = {r_{free} + {\left( {r_{bound} - r_{free}} \right)\frac{\begin{matrix} {\left( {K_{D} + {BLYS} + P} \right) -} \\ \sqrt{\left( {K_{D} + {BLYS} + P} \right)^{2} - {4 \cdot {BLYS} \cdot P}} \end{matrix}}{2 \cdot P}}}} \end{matrix}$

[0555] where:

[0556] r_(obs)=observed anisotrpy, r_(free)=anisotropy of free peptide, r_(bound)=anisotropy of bound peptide, K_(D)=dissociation constant, BLyS=total BLyS concentration, and P=total fluorescein labeled peptide concentration.

[0557] Binding reactions containing 50 nM fluorescein-labeled peptide and a varied concentration of BLyS in a volume between 10 and 20 μL per well were performed in 384 well microplates. Reactions were assayed using a Tecan Polarion fluorescence polarization plate reader. Cross-competition studies between peptides were performed using 50 nM fluorescein-labeled peptide and 1-2 μM BLyS in the presence and absence of 100 μM unlabeled peptide. The influence of pH on the observed K_(D) was investigated at pH 6.0 using the primary binding buffer [15 mM sodium citrate, 120 mM NaCl, 0.01% Tween 20] and at pH 9.0 using 200 mM sodium bicarbonate, 125 mM sodium chloride. Other buffers in which dissociation constants of BLyS Binding polypeptides were detremined include: [pH 6.0, 0.01% Tween], [pH 6.0, 0.1% gelatin], [pH5.0, 0.01% Tween], [pH9.0, 0.1% Tween], [pH6.0, 15% ethylene glycol, 0.01% Tween],], [pH5.0, 15% ethylene glycol, 0.01% Tween], and [pH9.0, 15% ethylene glycol, 0.01% Tween]. All six of the peptides (DX212, DX214, DX216, DX217, DX219, and DX221) bound BLyS in solution with approximately the same affinity (K_(D)=0.4-3 μM). Cross-competition studies demonstrated that all peptides compete with each other for BLyS binding, which suggests that they all bind to the same site on BLyS.

EXAMPLE 3

[0558] Chromatographic Screening of Immobilized BLyS Binding Polypeptides

[0559] A reversed phase analytical assay was used in the assessment of the chromatographic performance of the six affinity media.

[0560] The six affinity media (BLyS binding polypeptides bound to SEPHAROSE 4 Fast Flow) and a control column, (hydrolyzed NHS-SEPHAROSE 4 Fast Flow) were packed into 3×50 mm glass Omnifit columns (350 μl). All columns were tested at 200 μl/min (170 cm/hr) using a Watson/Marlow 101 ru peristaltic pump. This setup allowed free use of numerous wash, protein and elution conditions. Detection was made using a Waters 2487 UV/VIS detector at 214 nm and 280 nm connected to a Waters Millennium workstation.

[0561] Initial screens with purified BLyS at 30 μg/ml in PBS/0.01% Tween 20, pH 7.2 showed 65% recovery in the flow-through of the control column (1 ml, 30 μg total). However, it was immediately clear that all of the columns, when tested in the same manner, bound BLyS quantitatively from solution, but did not release the protein. The columns were then tested with an array of possible elution conditions and monitored at 214 nm and 280 nm for release of BLyS. Fractions indicating possible BLyS elution based on UV absorbance were collected and analyzed by reversed phase chromatography for confirmation. Conditions tested are shown in Table 10 (below). TABLE 10 Chromatographic Elution Conditions, BLyS Affinity Media Chaotropic Other Salts Orgainc Salts and pH Other Buffers and pH Solvents 1 M urea, 1 M NaCl, 30 mM H₃PO₄, 50% ethylene pH 7 pH 7 and 5 pH 2 glycol, pH 4 2 M urea 2 M NaCl, 200 mM NaCO₃, 50% ethylene pH 7 and 5 pH 10 glycol, pH 7 4 M urea, 2 M MgCl₂, 50-100 mM NaOAc, 20% ethanol pH 7 pH 7.6 pH 5 2 M guanidine, 2 M CaCl₂, 2 M imidazole, 50% ethanol pH 7 pH 6 pH 6 1 M citrate, 0.6 M Histidine, 18% butanol pH 6 pH 6 1 M Arginine, 30% glycerol pH6 100 mM EGTA, EDTA 1 M sorbitol, pH 7

[0562] Columns made with DX214, DX216, DX217, DX219 and DX221 affinity media would only release BLyS in the presence of pH 2 buffer (30 mM H₃PO₄/150 mM NaCl, pH 2). Recoveries from these columns ranged between 30% and 65%. In contrast, DX212 affinity media released BLyS with good recoveries on elution with 50% ethylene glycol, pH 4 (50% ethylene glycol/100 mM sodium acetate, pH 4) or with 50% ethylene glycol, pH 5 (50% ethylene glycol/100 mM sodium acetate, pH 5).

[0563] To determine if the elution conditions were effective at maintaining BLyS in its native trimeric form, size exclusion chromatography (SEC) was used to assay native BLyS and BLyS exposed to both pH 2 and 50% ethylene glycol/100 mM sodium acetate, pH 5. SEC analysis of BLyS following incubation at pH 2 revealed the presence of two new peaks, corresponding in size to apparent multimer and monomer forms, with no evidence of the native trimer. Incubation in 50% ethylene glycol resulted in 16% multimer, but otherwise maintained trimer. Later SEC results on material eluted from the DX212 column with 50% ethylene glycol (pH 5.0) did not show the multimer.

EXAMPLE 4

[0564] Capture of BLyS from Cell Culture Supernatants

[0565] The DX212, DX219, and DX214 affinity columns were tested for their ability to purify BLyS from cell culture supernatants. BLyS, at approximately 40 μg/ml, was spiked into thawed cell culture supernatants from CHO and Sf9 cell lines. Approximately 100 μg BLyS (2.5 ml total) was loaded onto each column. Levels of BLyS in Sf9 flow-through samples could not be determined in the reversed phase HPLC assay. BLyS was eluted with elution buffer [50% ethylene glycol, 100 mM NaAc, pH5.0]. BLyS protein recovery from these experiments ranged from 29.4% to complete recovery, with purities ranging from 76% to 96.5%.

EXAMPLE 5

[0566] Synthesis of Further BLyS Binding Peptides

[0567] Once a promising BLyS binding polypeptide has been isolated, improvements to that polypeptide can be made by changing, adding or removing individual or multiple amino acid residues from the polypeptide. Amino acid substitutions can be conservative or non conservative. Conservative amino acids exchanges include, for example, the exchange of aromatic residues (e.g., phenylalanine, tryptophan, and tyrosine) for one another, the exchange of hydrophobic residues (e.g, leucine, isoleucine, and valine) for one another, the exchange of polar residues (e.g., glutamine and asparagine) for one another, the exchange of acidic residues (e.g., arginine, lysine, and histidine) for one another, and the exchange of small residues (e.g., alanine, serine, threonine, methionine, and glycine) for one another, the exchange of aromatic residues for one another. Additionally, nonclassical amino acids, chemical amino acid analogs, or chemically modified classical amino acids can be introduced as a substitution or addition to a BLyS binding polypeptide of the invention. Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, 2,4-diaminobutyric acid (Dbu), 4-aminobutyric acid (bAbu), 2-aminobutyric acid (Abu), 6-amino hexanoic acid (epsilon-Ahx), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (bAib), 3-aminopropanoic acid (bAla), ornithine (Orn), norleucine (Nle), norvaline (Nva), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), sarcosine (MeGly), citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs in general. By way of example, four modified peptides based on the DX212 sequence have been designed:

[0568] 1. Ac-AGK(Ac)EPCYFYWECAVSGPGPEGGGK (SEQ ID NO: 169)—internal lysine side chain acetylated;

[0569] 2. Ac-AGREPCYFYWECAVSGPGPEGGGK (SEQ ID NO: 170)—arginine substitution;

[0570] 3. Ac-AGQEPCYFYWECAVSGPGPEGGGK (SEQ ID NO: 171)—glutamine substitution;

[0571] 4. Ac-AGNleEPCYFYWECAVSGPGPEGGGK (SEQ ID NO: 172)—norleucine substitution.

EXAMPLE 6

[0572] Affinity Maturation of BLyS Binding Polypeptides

[0573] In order to identify high affinity BLyS-binding polypeptides, a BLyS Affinity Maturation Library (BAML) was designed around a 14-mer linear peptide template sequence having fixed amino acid residues at 5 of the 14 positions. 3 of the 5 fixed residues corresponded to a highly conserved DxLT tetrapeptide amino acid motif (SEQ ID NO: 446) isolated from both the constrained and linear peptide libraries. The design of the 14-mer allowed for some amino acid variation at each of the remaining 9 positions, however, preference was given for a particular amino acid at each of these positions. Analysis of binding affinity of the newly isolated peptides for BLyS was evaluated by direct and indirect phage ELISA and fluorescence anisotropy.

[0574] BAML was designed on a 14-mer linear (non-constrained) template peptide sequence having fixed residues at positions 1 (Ala), 5 (Asp), 7 (Leu), 8 (Thr), and 10 (Leu). The amino acid sequence of positions 3-14 in the BAML template most closely resembles a binding polypeptide isolated from the PhD 12 linear polypeptide library (see Table 7, supra). Residues An at position 1 (fixed Ala) and position 2 (variable) were included to extend the length and presentation of the BLyS-binding sequence. Positions 5-8 correspond to the DxLT motif found in peptide isolates from both the constrained and linear peptide libraries (see Tables 1-8, supra). Since hydrophobic amino acids (L, M, I, A, and G) were found at position 10 in 85% of the original isolates, a Leu residue, occurring in 42% of the isolates, was fixed at that position in the BAML template peptide.

[0575] Table 11 shows the design of the 14-mer BAML template sequence. TABLE 11 BAML template sequence (14-mer) SEQ ID amino acid position NO: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 A n w y D s L T k L w l p d 184

[0576] Referring to Table 11, the upper case letters indicate the fixed residues at positions 1, 5, 7, 8, and 10 of the template. Lower case letters designate preferred amino acids at those positions, however the design of the variegated DNA template encoding the 14-mer allows for some sequence variation at these positions.

[0577] Table 12 shows the design of the variegated DNA template used to generate the BAML peptides. TABLE 12 BAML DNA template sequence (14-mer) codon position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 codons* GCT eez zjj zez GAT zqz CTT ACT eej CTC zjj qzz qqz jez

[0578] Referring to Table 12, the nucleotide coding sequences for the fixed amino acids in the BAML 14-mer template are shown in upper case letters. The letters “e”, “j”, “q”, and “z” in the variegated DNA template each represent a particular mixture of nucleoside bases present in the input dNTPs for each position:

[0579] j=79% guanine, 7% cytosine, 7% adenine, 7% thymine

[0580] q=7% guanine, 79% cytosine, 7% adenine, 7% thymine

[0581] e=7% guanine, 7% cytosine, 79% adenine, 7% thymine

[0582] z=7% guanine, 7% cytosine, 7% adenine, 79% thymine.

[0583] The codons of the DNA template were designed to skew the encoded variable amino acid toward the preferred amino acid at each position shown in SEQ ID NO: 184 (Table 11, lower case). Later sequencing of phage isolates showed that, at any particular position, preferred residues occurred at a frequency of from 44% to 70%.

[0584] Synthetic DNA sequences fitting the DNA template were amplified by large scale PCR. The amplified DNAs were restriction digested for insertion into a M13 phage expression vector (MANP vector, Dyax Corp., Cambridge, Mass.), and vectors bearing the inserts were used to transform M13 phage by electroporation, to produce the BAML.

[0585] Recombinant phage were collected and purified by PEG precipitation and titered. A total of 3.2×10¹³ PFU were amplified in BAML from 1.6×10⁹ transformants.

[0586] Screening BAML

[0587] As outlined in Table 13, a two-step competition method, starting with the original BAML library, was used over 4 rounds of screening to isolate the highest affinity BLyS-binding polypeptides from the BAML. Prior to screening, the amplified BAML was contacted with Seradyn streptavidin-coated magnetic beads (MG-SA, Seradyn, Indianapolis, Ind.), to remove bead- and streptavidin-binding phage.

[0588] For screening BAML, phage were incubated in solution with biotinylated BLyS (b-BLyS) in 200 μl PBS, pH 7.4, Tween-20 (0.1%), to form phage/b-BLyS binding complexes. For the first competition step, unlabeled BLyS (1-2 μM) was added to the phage/b-BLyS binding complex mixture in solution and incubated for 1-20 hrs. (See Table 13.) The phage/b-BLyS complexes remaining in solution after incubation with unlabeled BLyS were captured by brief (10 min. on rotator) incubation with MG-SA streptavidin beads (50 μl). After capture of the phage/b-BLyS complexes on streptavidin beads, the unbound fraction was removed and beads were washed 15-20 times with 1 ml PBS-Tween 20 prior to the second competition step. The phage/unlabeled BLyS complexes from the round 1 competition step only, were collected and used as a fraction of the input phage for the second round of screening along with the bead-captured phage/b-BLyS complexes, however, in each subsequent round of screening only the bead-associated phage were collected after the first competition step for further screening, and the phage/unlabeled BLyS complexes were discarded.

[0589] For the second competition step, the competitor peptide was a polypeptide (DX221; SEQ ID NO: 168) based on a BLyS-binding polypeptide isolated from the PhD 12 library in the initial screenings described above. The phage/b-BLyS complexes bound to streptavidin, collected after the first competition incubation step, were serially diluted with 50 μM DX221 BLyS-binding peptide (K_(D)=3 μM) in 300 μl PBS-Tween-20 (0.1%). A series of short incubations (3-4 per round, for 1 hour) of the phage/b-BLyS complexes with DX221 followed by a final incubation of from overnight (O/N, for rounds 1, 2, and 4) to 3 days (for round 3). (See Table 13.) The second competition step in round 4 included an incubation with 67 nM BLyS for 1 hour prior to incubation with DX221. The streptavidin bead-associated phage/b-BLyS binding complexes remaining after the DX221 competition step in round 4 were collected for further analysis. TABLE 13 BLyS affinity maturation library (BAML) screening conditions First Second Competition Competition Screening Input Incubation Competitor Incubation Peptide Round phage¹ b-BLyS² Time (hrs) (BLyS) Time (hrs) Elutions 1 1.5 × 10¹¹ 100 nM 2 2 μM 1 50 μM DX221, 4 × 1 hr, then O/N 2   2 × 10¹⁰ 100 nM 1 1 μM 20 50 μM DX221, 3 × 1 hr, then O/N 3 6.5 × 10¹⁰ 100 pM 16 1 μM 3 50 μM DX221 4 × 1 hr, then 3 days 4 6.0 × 10¹⁰  10 pM 16 1 μM 2 67 nM BLyS, 1 hr; 50 μM DX221 + 67 nM BLyS 3 × 1 hr, O/N, then add'1 4 hrs

[0590] ELISA Analysis

[0591] Approximately four hundred BAML isolates from rounds 2, 3 and 4 of the above screening were analyzed by direct and indirect phage ELISA assays.

[0592] For indirect phage ELISA, Immulon-2HB plates (Dynex Technologies, Inc., Chantilly, Va.) were coated with 100 μl of 1 μg/ml Immunopure streptavidin (Pierce, Rockford, Ill.) diluted in PBS. 100 μl of a series of 10-fold dilutions of b-BLyS (0-0.1 μg/ml in PBS) were immobilized in the streptavidin-coated wells (1 hr, 37° C.). After washing, 1-25 μl of overnight culture of E. coli infected with the individual phage plaques were added to the appropriate wells and incubated for 1 hour, followed by 10 washes with PBS-Tween-20. Anti-M13 antibody conjugated to horseradish peroxidase (1:10,000 in PBS-Tween-20) was added to the wells (30 min., room temperature), the color reagent TMB was used and the plates read at OD 630 nm.

[0593] Individual phage isolates binding to immobilized BLyS were sequenced and the sequences analyzed. The unique sequences of the BAML BLyS-binding 14-mer display peptides are shown in Table 14.

[0594] Analysis of the peptides reveals a significant sequence “collapse” around one motif: W₃YDPLTKLWL₁₂ (SEQ ID NO: 436) (subscripts indicate amino acid position in the 14-mer display peptide sequence). This most numerous core motif includes the four fixed residues from the original BAML template, i.e., Asp (D) at position 5, Leu (L) at position 7, Thr (T) at position 8, and Leu (L) at position 10. In addition, 5 of the 6 preferred residues from the original BAML template sequence were included in this motif (see Table 11).

[0595] 73% (143 of 197) of the round 4 isolates included this core motif (SEQ ID NO: 436). Single residue substitutions within the 10-mer core motif centered on positions 4 (Y→F) and 12 (L→F, I, or V), with the substitutions at position 12 being alternative hydrophobic residues for Leu.

[0596] For the three remaining variable positions (i.e., 2, 13, and 14), selection was not as stringent, although some preferences were apparent, being either built into the library or persisting through rounds of selection. For example, in round 4 isolates, 51% included Asn at position 2; 77% included Pro at position 13; and 32% included Asp at position 14. The presence of Val (27%) or Glu (19%) at position 14 was among the most highly selected in the round 4 isolates, in comparison to their theoretical proportion (4% each) at position 14 in BAML.

[0597] The sequences in Table 14 are grouped according to their degree of difference from the core sequence (SEQ ID NO: 436). TABLE 14 Sequences of BAML Phage Isolates (from Rounds 2, 3, 4) 14-mer amino acid position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 SEQ ID NO: A n w y D s L T k L w l p d consensus; 184 A N W Y D P L T K L W L P D 186 A N W Y D P L T K L W L P E 187 A N W Y D P L T K L W L P G 188 A N W Y D P L T K L W L P V 189 A N W Y D P L T K L W L S D 190 A N W Y D P L T K L W L N D 191 A N W Y D P L T K L W L P T 192 A N W Y D P L T K L W L P A 193 A N W Y D P L T K L W L P N 194 A N W Y D P L T K L W L V D 195 A N W Y D P L T K L W L H D 196 A N W Y D P L T K L W L T D 197 A N W Y D P L T K L W L P H 198 A N W Y D P L T K L W L T V 199 A N W Y D P L T K L W L L D 200 A N W Y D P L T K L W L L E 201 A N W Y D P L T K L W L H E 202 A N W Y D P L T K L W L P R 203 A N W Y D P L T K L W L A D 204 A N W Y D P L T K L W L P Y 205 A N W Y D P L T K L W L P I 206 A N W Y D P L T K L W L I D 207 A N W Y D P L T K L W L R D 208 A Y W Y D P L T K L W L P D 209 A Y W Y D P L T K L W L L E 210 A Y W Y D P L T K L W L R V 211 A Y W Y D P L T K L W L P E 212 A Y W Y D P L T K L W L P V 213 A Y W Y D P L T K L W L H Q 214 A Y W Y D P L T K L W L P A 215 A Y W Y D P L T K L W L R V 216 A Y W Y D P L T K L W L P G 217 A Y W Y D P L T K L W L R Y 218 A Y W Y D P L T K L W L P Y 219 A Y W Y D P L T K L W L L Y 220 A Y W Y D P L T K L W L R D 221 A Y W Y D P L T K L W L P V 222 A Y W Y D P L T K L W L L G 223 A Y W Y D P L T K L W L T H 224 A Y W Y D P L T K L W L P T 225 A Y W Y D P L T K L W L L V 226 A Y W Y D P L T K L W L Y Y 227 A Y W Y D P L T K L W L S D 228 A S W Y D P L T K L W L P A 229 A S W Y D P L T K L W L H D 230 A S W Y D P L T K L W L P G 231 A S W Y D P L T K L W L P Q 232 A S W Y D P L T K L W L P Y 233 A S W Y D P L T K L W L P H 234 A S W Y D P L T K L W L P V 235 A S W Y D P L T K L W L P I 236 A S W Y D P L T K L W L P E 237 A F W Y D P L T K L W L R V 238 A F W Y D P L T K L W L P E 239 A F W Y D P L T K L W L L E 240 A F W Y D P L T K L W L P V 241 A I W Y D P L T K L W L P E 242 A I W Y D P L T K L W L P D 243 A I W Y D P L T K L W L H D 244 A I W Y D P L T K L W L T D 245 A I W Y D P L T K L W L P F 246 A I W Y D P L T K L W L L D 247 A I W Y D P L T K L W L P R 248 A I W Y D P L T K L W L P A 249 A I W Y D P L T K L W L T A 250 A I W Y D P L T K L W L A V 251 A I W Y D P L T K L W L P G 252 A I W Y D P L T K L W L R V 253 A I W Y D P L T K L W L P H 254 A I W Y D P L T K L W L R E 255 A I W Y D P L T K L W L S D 256 A T W Y D P L T K L W L P A 257 A T W Y D P L T K L W L A D 258 A T W Y D P L T K L W L T S 259 A T W Y D P L T K L W L P G 260 A T W Y D P L T K L W L P Y 261 A T W Y D P L T K L W L S G 262 A T W Y D P L T K L W L P V 263 A T W Y D P L T K L W L P D 264 A D W Y D P L T K L W L P V 265 A D W Y D P L T K L W L P K 266 A D W Y D P L T K L W L P D 267 A D W Y D P L T K L W L P E 268 A D W Y D P L T K L W L H Q 269 A E W Y D P L T K L W L R D 270 A E W Y D P L T K L W L P D 271 A E W Y D P L T K L W L P Y 272 A L W Y D P L T K L W L P A 273 A L W Y D P L T K L W L P D 274 A L W Y D P L T K L W L R G 275 A L W Y D P L T K L W L L G 276 A M W Y D P L T K L W L P A 277 A M W Y D P L T K L W L Q V 278 A M W Y D P L T K L W L L G 279 A A W Y D P L T K L W L P D 280 A A W Y D P L T K L W L A D 281 A A W Y D P L T K L W L L D 282 A H W Y D P L T K L W L T D 283 A H W Y D P L T K L W L P V 284 A H W Y D P L T K L W L H D 285 A H W Y D P L T K L W L P D 286 A P W Y D P L T K L W L H D 287 A P W Y D P L T K L W L P V 288 A Q W Y D P L T K L W L P E 289 A Q W Y D P L T K L W L P Y 290 A Q W Y D P L T K L W L P R 291 A K W Y D P L T K L W L P D 292 A K W Y D P L T K L W L P V 293 A K W Y D P L T K L W L P V 294 A K W Y D P L T K L W L N G 295 A W W Y D P L T K L W L P A 296 A V W Y D P L T K L W L T D 297 A N W Y D P L T K L W L P D 186 A Y E Y D P L T K L W L L Y 298 A T K Y D P L T K L W L P D 299 A T L Y D P L T K L W L P G 300 A I R Y D P L T K L W L P Y 301 A E R Y D P L T K L W L P H 302 A D R Y D P L T K L W L P Q 303 A N S Y D P L T K L W L P E 304 A I L Y D P L T K L W L P D 305 A N W Y D P L T K L W L P D 186 A N W F D P L T K L W L P Q 306 A N W F D P L T K L W L P V 307 A N W F D P L T K L W L T D 308 A N W F D P L T K L W L P D 309 A N W F D P L T K L W L P G 310 A N W F D P L T K L W L P E 311 A N W F D P L T K L W L P A 312 A N W F D P L T K L W L P N 313 A N W F D P L T K L W L S E 314 A N W F D P L T K L W L H D 315 A N W F D P L T K L W L V D 316 A Y W F D P L T K L W L P D 317 A Y W Y D P L T K L W L P V 318 A Y W F D P L T K L W L P A 319 A Q W F D P L T K L W L P D 320 A H W F D P L T K L W L P D 321 A T W Y D P L T K L W L P V 322 A N W Y D P L T K L W L P D 186 A Y W Y D P L T K L W L P V 323 A Y W Y D S L T K L W L H D 324 A N W Y D S L T K L W I P D 325 A N W Y D S L T K L W L P V 326 A N W Y D S L T K L W L P D 327 A N W Y D S L T K L W L A D 328 A N W Y D S L T K L W L P A 329 A N W Y D S L T K L W L Y E 330 A N W Y D P L T K L W L P D 186 A G W Y D S L T K L W L P D 331 A V W Y D S L T K L W L T D 332 A N W Y D A L T K L W L P V 333 A Y W Y D T L T K L W L P N 334 A N W Y D P L T K L W L P D 186 A F W Y D P L T N L W L L E 335 A Y W Y D P L T G L W L L G 336 A Y W Y D P L T G L W L L Y 337 A Y W Y D P L T G L W L R V 338 A Y W Y D P L T E L W L R L 339 A N W Y D P L T K L W L P D 186 A M W Y D P L T K L S L P D 340 A Y W Y D P L T K L S L L V 341 A I W Y D P L T K L S L T V 342 A I W Y D P L T K L S L L V 343 A D W Y D P L T K L S L L L 344 A Y W Y D P L T K L R L L E 345 A D W Y D P L T K L 4 L L V 346 A D W Y D P L T K L R L I V 347 A I W Y D P L T K L Y L P D 348 A I W Y D P L T K L G L L V 349 A N W Y D P L T K L T L L V 350 A N W Y D P L T K L L L P N 351 A N W Y D P L T K L W L P D 186 A S W Y D P L T K L W F P D 352 A N W Y D P L T K L W F P D 353 A N W Y D P L T K L W F S D 354 A S W Y D P L T K L W F P V 355 A D W Y D P L T K L W F P V 356 A S W Y D P L T K L W F P K 357 A K W Y D P L T K L W F P D 358 A S W Y D P L T K L W F L E 359 A N W Y D P L T K L W F P A 360 A T W Y D P L T K L W F P D 361 A I W Y D P L T K L W F P E 362 A I W Y D P L T K L W F P D 363 A I W Y D P L T K L W F P G 364 A Y W Y D P L T K L W F P H 365 A N W Y D P L T K L W F P V 366 A Y W Y D P L T K L W F P D 367 A G W Y D P L T K L W F P D 368 A I W Y D P L T K L W F P T 369 A K W Y D P L T K L W F P A 370 A Y W Y D P L T K L W F F D 371 A N W Y D P L T K L W F A D 372 A N W Y D P L T K L W L P D 186 A N W Y D P L T K L W F P Y 373 A D W Y D P L T K L W F R D 374 A N W Y D P L T K L W V P D 375 A D W Y D P L T K L W V P A 376 A N W Y D P L T K L W V P N 377 A N W Y D P L T K L W V P E 378 A N W Y D P L T K L W V P Q 379 A E W Y D P L T K L W V P K 380 A Q W Y D P L T K L W V P V 381 A N W Y D P L T K L W V P Y 382 A L W Y D P L T K L W V P Y 383 A N W Y D P L T K L W V P G 384 A S W Y D P L T K L W I P Y 385 A D W Y D P L T K L W I P G 386 A N W Y D P L T K L W I P Y 387 A KW Y D P L T K L W I P Y 388 A I W Y D P L T K L W I P N 389 A T W Y D P L T K L W I P Q 390 A N W Y D P L T K L W L P D 186 A S W Y D P L T N L W V P D 391 A Y E Y D P L T N L W L L Y 392 A Y W Y D P L T N L S L L V 393 A Y W Y D P L T K L S I L E 394 A N W Y D S L T K L W I P Y 395 A H W F D P L T Q L K I R V 396 A Y W C D P L T K L C I L E 397 A N S Y D P L T K L W F P Y 398 A N L Y D P L T K L W V P Y 399 A N W Y D P L T K L W L H D 400 A N W Y D S L T K L W F P D 401 A T S Y D S L T K L W L P A 402 A C W Y D S L T K L C H R E 403 A I G N D P L T K L W I P Y 404 A N W Q D C L T K L C L A G 405 A Y W F D P L T N L W L L E 406 A Y W Y D P L T N L S L L V 407 A N C F D S L T R L W L C D 408 A C A Y D A L T K L C L P A 409 A N W Y D P L T N L S L L L 410 A Y W Y D P L T Q L S L L V 411 A Y R Y D A L T G L W L L Y 412 A Y W N D P L T K L K L R L 413 A Y W Y D P L T Q L S L L V 414 A Y R Y D A L T G L W L L Y 415 A Y R Y D S L T N L W L L Y 416 A Y W Y D P L T K L S I L E 417 A S C Y D P L T K L C F P V 418 A F W F D P L T G L W L L E 419 A N W Y D P L T K L W L P D 186 A H W Y D P L T K L S I R V 420 A P W Y D S L T K L W F P S 421 A N C Y D T L T K L W L T C 422 A N W Y D S L T K L S L P D 423 A Y A Y D F L T Q L S L P D 424 A F R Y D S L T G L W L R Y 425 A N C Y D S L T K L W L P C 426 A N G Y D L L T N L S V S D 427 A N W Y D P L T R L W I P V 428 A L K F D Y L T K L W L P D 429 A Y R Y D S L T K L W L P G 430 A Y C Y D S L T K L W I P D 431 A S W E D S L T K L W L S K 432 A Y W Y D S L T G L S L L V 433 A Y W Y D P L T Y L R L R V 434 A K C Y D S L T N L W L C D 435

[0598] Nearly all of the ELISA signals of the BAML isolates were higher than those isolated in the initial screen (see Example 1). For comparison, peptide 453-01-B07 (SEQ ID NO: 31) (K_(D)=700 nM) was used as a reference (positive control). Negative control MAEX (M13 phage with no insert) did not bind b-BLyS at any concentration tested.

[0599] For direct phage ELISA, the signal measured is a reflection of the ability of a set number of phage to bind to various concentrations of b-BLyS. Peptides tested by the direct phage ELISA assay were chosen based on high affinity for BLyS as determined in the indirect phage ELISA assay. For this assay, Immulon-2HB plates were coated with 0 or 1000 ng anti-Fd antibody (Sigma, St. Louis, Mo.). After washing (PBS-Tween-20), phage dilutions were added to saturate the available antibody and incubated for 1 hour, washed, then incubated with 100 μl of 10-fold dilutions of b-BLyS (0-1 μg/ml) for 1 hour at room temperature. Streptavidin-HRP (1:1000 in PBS-tween-20; Endogen, Woburn, Mass.) was added to the wells and incubated for 1 hour, developed using TMB and reading at OD 630 nm.

[0600] Determination of BAML Peptide K_(D) by Fluoresence Anisotropy.

[0601] Several peptides containing the 10-mer core structural motif or single-position variants of that motif identified by sequence analysis were synthesized with a short Gly-Gly-Lys linker sequence and the C-terminal lysine was labeled with fluorescein. These peptides, shown in Table 15, below, were synthesized by solid phase synthesis for determination of dissociation constant with respect to BLyS. The DX815 and DX876 polypeptides were derived from DX814 (SEQ ID NO: 186) by deletion of two N-terminal amino acids or the two amino acids N-terminal and C-terminal to the core peptide at (positions 3-12). DX816, DX817, DX819, and DX822 correspond to other BAML isolates (SEQ ID NOs: 189, 309, 353, 327, respectively). DX818 corresponds to isolate SEQ ID NO: 340, except that Asn has been substituted for Met at position 2. The K_(D) of several BLyS binding BAML peptides was determined by fluorescence anisotropy, performed as previously described. The sequence of DX822 without the -GGK linker (see SEQ ID NO: 327) matches the BAML template sequence (see Table 11). The BAML consensus sequence found in DX822 resulted in a more than 10-fold improvement in binding affinity for BLyS, as compared to one of the highest affinity binders isolated in the initial screen (453-01-B07, SEQ ID NO: 31). TABLE 15 Dissociation Constants of Synthetic BLyS-binding Polypeptides SEQ Peptide Sequence ID NO: K_(D) (nM) DX814 Ac-ANWYDPLTKLWLPDGGK-fitc 437 26 ±7  DX815 Ac-WYDPLTKLWLPDGGK-fitc 438 31 ± 13 DX876 Ac-WYDPLTKLWLGGK-fitc 439 171 ± 90  DX816 Ac-ANWYDPLTKLWLPVGGK-fitc 440 44 ± 15 DX817 Ac-ANWFDPLTKLWLPDGGK-fitc 441 32 ± 26 DX818 Ac-ANWYDPLTKLSLPDGGK-fitc 442 342 ± 108 DX819 Ac-ANWYDPLTKLWFPDGGK-fitc 443 69 ± 38 DX822 Ac-ANWYDSLTKLWLPDGGK-fitc 444 79 ± 54

[0602] Analysis of the BAML isolates revealed a lack of sequence conservation at position 2 (varied in the BAML template, see Table 11). To examine whether the N-terminal residues at positions 1 and 2 in the BAML sequence were necessary for binding to BLyS, a truncated version of DX814 comprising only residues 3-14 (DX815; see Table 15) was synthesized and analyzed by fluorescence anisotropy. The K_(D) for DX815 was indistinguishable from that of DX814, suggesting that residues 1-2 are not required for high affinity binding to BLyS. Further truncation of DX814 to the minimal core (residues 1-10, DX876) increased the K_(D) to 171 nM, indicating a contribution from Pro at position 13 and/or Asp at position 14 of the 14-mer to high affinity BLyS binding. Substitution of Val in DX816 at that position had little effect on the K_(D) (see Table 15). In comparing the BLyS-binding polypeptide DX221 (Ac-WTDSLTGLWFPDGGPGPEGGGK; K_(D)=3 μM; SEQ ID NO: 168) with the BAML peptide closest in sequence (DX819, Ac-ANWYDPLTKLWFPDGGK; K_(D)=69 nM; SEQ ID NO: 443), differences are seen at three positions 4 (T→Y), 6 (S→P), and 9 (G→K), indicating the contribution of these residues in binding affinity.

[0603] The synthesized BAML peptides exhibited K_(D) values in the low nanomolar range, two orders of magnitude lower than primary isolate-derived peptides (see Example 1). Phenylalanine substitutions (F₄→Y₄; F₁₂→L₁₂; Table 17) were the most common minor variations to the core sequence and these changes failed to significantly affect the dissociation constants of the synthesized peptides. A change at position 11 (W₁₁→S₁₁; DX818), however, resulted in an approximately 10-fold decrease in affinity compared to DX814.

[0604] Following the foregoing description, the characteristics important for affinity binding polypeptides permitting detection or separation of BLyS or BLyS-like polypeptides (BLyS target protein) in or from any solution can be appreciated. Additional binding polypeptide embodiments of the invention and alternative methods adapted to a particular solution or feed stream will be evident from studying the foregoing description. For instance, any spacer or linker sequences associated with BLyS binding polypeptides discussed above may be removed or substituted to yield additional BLyS binding polypeptides of this invention. Also, very high affinity polypeptide BLyS target binders suitable for in vivo therapeutic applications may be prepared, e.g., by selecting among the peptides isolated from the BAML, by selecting similar polypeptides under similarly stringent conditions from BAML or other peptide library, or by designing a polypeptide binding molecule following the descriptions above, e.g., of important structural motifs contributing to BLyS binding properties. All such embodiments and obvious alternatives are intended to be within the scope of this invention, as defined by the claims that follow.

[0605] The publications referred to above are hereby incorporated by reference in their entireties.

1 458 1 13 PRT Artificial Sequence BLyS binding polypeptide 1 Xaa Xaa Xaa Cys Xaa Pro Xaa Thr Gly Cys Xaa Xaa Xaa 1 5 10 2 14 PRT Artificial Sequence BLyS binding polypeptide 2 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 3 15 PRT Artificial Sequence BLyS binding polypeptide 3 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 15 4 16 PRT Artificial Sequence BLyS binding polypeptide 4 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 15 5 18 PRT Artificial Sequence BLyS binding polypeptide 5 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 15 Xaa Xaa 6 12 PRT Artificial Sequence BLyS binding polypeptide 6 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 7 13 PRT Artificial Sequence BLyS binding polypeptide 7 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 8 7 PRT Artificial Sequence BLyS binding polypeptide 8 Cys Xaa Pro Xaa Thr Gly Cys 1 5 9 8 PRT Artificial Sequence BLyS binding polypeptide 9 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 9 PRT Artificial Sequence BLyS binding polypeptide 10 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 11 10 PRT Artificial Sequence BLyS binding polypeptide 11 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 12 12 PRT Artificial Sequence BLyS binding polypeptide 12 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 13 8 PRT Artificial Sequence c-terminal linker 13 Pro Gly Pro Glu Gly Gly Gly Lys 1 5 14 12 PRT Artificial Sequence phage display library template 14 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 15 13 PRT Artificial Sequence phage display library template 15 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 16 14 PRT Artificial Sequence phage display library template 16 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 17 15 PRT Artificial Sequence phage display library template 17 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 15 18 16 PRT Artificial Sequence phage display library template 18 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 15 19 18 PRT Artificial Sequence phage display library template 19 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 15 Xaa Xaa 20 12 PRT Artificial Sequence BLyS binding polypeptide 20 His Leu Arg Cys Trp Ser Thr Asn Cys Arg Tyr Asp 1 5 10 21 12 PRT Artificial Sequence BLyS binding polypeptide 21 Val Met Asp Cys Leu Ile Asn Arg Cys Asp Thr Val 1 5 10 22 13 PRT Artificial Sequence BLyS binding polypeptide 22 Lys Ser Lys Cys Phe Phe Pro Trp Glu Cys Gln Gln Ala 1 5 10 23 13 PRT Artificial Sequence BLyS binding polypeptide 23 Ala Met Lys Cys Tyr Phe Pro Trp Glu Cys Ala Asn Gly 1 5 10 24 14 PRT Artificial Sequence BLyS binding polypeptide 24 Glu Asn Val Ala Cys Tyr Phe Pro Trp Glu Cys His His Pro 1 5 10 25 13 PRT Artificial Sequence BLyS binding polypeptide 25 Asn Ala Pro Cys Tyr Phe Pro Trp Glu Cys Phe Ser Ile 1 5 10 26 13 PRT Artificial Sequence BLyS binding polypeptide 26 Ser Val Asn Cys Trp Phe Pro Trp Glu Cys Val Gly Asn 1 5 10 27 13 PRT Artificial Sequence BLyS binding polypeptide 27 Lys Glu Pro Cys Tyr Phe Tyr Trp Glu Cys Ala Val Ser 1 5 10 28 14 PRT Artificial Sequence BLyS binding polypeptide 28 Asp Thr Asn Cys Asp Leu Leu Thr Lys Met Cys Gly Pro Gln 1 5 10 29 14 PRT Artificial Sequence BLyS binding polypeptide 29 Gly Thr Pro Cys Asp Leu Leu Thr Lys Leu Cys Leu Leu Trp 1 5 10 30 14 PRT Artificial Sequence BLyS binding polypeptide 30 Met Ser Glu Cys Asp Leu Leu Thr Lys Ile Cys Leu Met Gly 1 5 10 31 14 PRT Artificial Sequence BLyS binding polypeptide 31 Val Pro Phe Cys Asp Leu Leu Thr Lys His Cys Phe Glu Ala 1 5 10 32 14 PRT Artificial Sequence BLyS binding polypeptide 32 Val Pro Phe Cys Asp Leu Leu Thr Lys His Cys Phe Glu Ala 1 5 10 33 14 PRT Artificial Sequence BLyS binding polypeptide 33 Trp Ser Ala Cys Asp Leu Leu Thr Lys Gln Cys Val Gln Val 1 5 10 34 13 PRT Artificial Sequence BLyS binding polypeptide 34 Asp Gly Cys Asp Glu Leu Thr Lys Ile Cys Gly Met Lys 1 5 10 35 14 PRT Artificial Sequence BLyS binding polypeptide 35 Lys Ser Trp Cys Asp Glu Leu Thr Lys Val Cys Phe Asp Pro 1 5 10 36 14 PRT Artificial Sequence BLyS binding polypeptide 36 Lys Trp Met Cys Asp Glu Leu Thr Lys Gln Cys Gln Tyr Val 1 5 10 37 14 PRT Artificial Sequence BLyS binding polypeptide 37 Met Lys Tyr Cys Asp Glu Leu Thr Lys Ile Cys Val Gly Trp 1 5 10 38 14 PRT Artificial Sequence BLyS binding polypeptide 38 Tyr Phe Gln Cys Asp Glu Leu Thr Lys Met Cys Trp Gln Lys 1 5 10 39 14 PRT Artificial Sequence BLyS binding polypeptide 39 Ala Met His Cys Asp Lys Leu Thr Lys His Cys Lys Phe His 1 5 10 40 13 PRT Artificial Sequence BLyS binding polypeptide 40 Val Pro Tyr Cys Asp Lys Leu Thr Lys Ile Cys Gln Trp 1 5 10 41 14 PRT Artificial Sequence BLyS binding polypeptide 41 Glu Val Phe Cys Asp Val Leu Thr Lys Val Cys Phe His Asp 1 5 10 42 14 PRT Artificial Sequence BLyS binding polypeptide 42 Lys Pro Lys Cys Asp Val Leu Thr Lys Met Cys Asp Trp Leu 1 5 10 43 14 PRT Artificial Sequence BLyS binding polypeptide 43 Thr Gln His Cys Asp Val Leu Thr Lys Gln Cys Phe Thr Ile 1 5 10 44 14 PRT Artificial Sequence BLyS binding polypeptide 44 Gly His Phe Cys Asp Arg Leu Thr Lys Tyr Cys Phe Glu Pro 1 5 10 45 14 PRT Artificial Sequence BLyS binding polypeptide 45 His Ile Gln Cys Asp Arg Leu Thr Lys Ser Cys Leu Ser Val 1 5 10 46 14 PRT Artificial Sequence BLyS binding polypeptide 46 Ile Lys Ala Cys Asp Ile Leu Thr Lys Val Cys Trp Pro Pro 1 5 10 47 14 PRT Artificial Sequence BLyS binding polypeptide 47 Gln Phe Asp Cys Asp Pro Leu Thr Lys Tyr Cys Gly Glu Phe 1 5 10 48 14 PRT Artificial Sequence BLyS binding polypeptide 48 Lys Met Tyr Cys Asp His Leu Thr Gly Tyr Cys Trp Pro Glu 1 5 10 49 14 PRT Artificial Sequence BLyS binding polypeptide 49 Met Gln Ser Cys Asp Ile Leu Thr Gly Tyr Cys Phe Lys Arg 1 5 10 50 14 PRT Artificial Sequence BLyS binding polypeptide 50 Gly Pro Trp Cys Asp Ile Leu Thr Gly Phe Cys Leu Ala Gln 1 5 10 51 14 PRT Artificial Sequence BLyS binding polypeptide 51 Ser Val Arg Cys Asp Leu Leu Thr Gly Trp Cys Pro Val Trp 1 5 10 52 14 PRT Artificial Sequence BLyS binding polypeptide 52 Pro Ala Asp Cys Asp Pro Leu Thr Asn Ile Cys Phe Trp Lys 1 5 10 53 14 PRT Artificial Sequence BLyS binding polypeptide 53 Thr Asn Val Cys Asp Pro Leu Thr Asn Val Cys Phe Met Asn 1 5 10 54 14 PRT Artificial Sequence BLyS binding polypeptide 54 Glu His Trp Cys Asp Asp Leu Thr His Leu Cys Phe Arg Leu 1 5 10 55 14 PRT Artificial Sequence BLyS binding polypeptide 55 Gly Tyr Trp Cys Asp Val Leu Thr Asn Asn Cys Trp Lys Ile 1 5 10 56 14 PRT Artificial Sequence BLyS binding polypeptide 56 Leu Tyr Asn Cys Asp Tyr Leu Thr Arg Leu Cys Phe Glu Pro 1 5 10 57 14 PRT Artificial Sequence BLyS binding polypeptide 57 His Val Asp Cys Leu Leu His Pro Lys Ala Cys Tyr Lys Tyr 1 5 10 58 14 PRT Artificial Sequence BLyS binding polypeptide 58 Val Gln Asp Cys Leu Leu His Pro Lys Ala Cys Gln Met Gln 1 5 10 59 14 PRT Artificial Sequence BLyS binding polypeptide 59 Lys Phe Asp Cys Leu Leu Lys Pro Met Phe Cys Ser Asn His 1 5 10 60 14 PRT Artificial Sequence BLyS binding polypeptide 60 Phe Ala Asp Cys Leu Ile His Pro Lys Ser Cys Lys Pro Leu 1 5 10 61 14 PRT Artificial Sequence BLyS binding polypeptide 61 His Gly Asn Cys Tyr Pro Phe Pro Trp Glu Cys Glu Ser Lys 1 5 10 62 14 PRT Artificial Sequence BLyS binding polypeptide 62 Met Ile Ile Val Leu Leu Leu Leu Arg Phe Ala Ile Ser Arg 1 5 10 63 14 PRT Artificial Sequence BLyS binding polypeptide 63 Ser Leu Leu Val Ile Phe Leu Leu Ile Gly Ala Gly Ser Leu 1 5 10 64 15 PRT Artificial Sequence BLyS binding polypeptide 64 Phe His Pro Cys Asp Met Leu Thr Gly Ile Trp Cys Gln Pro Asn 1 5 10 15 65 15 PRT Artificial Sequence BLyS binding polypeptide 65 Ser Lys Arg Cys Asp Leu Leu Thr Lys Met Trp Cys Glu Thr Glu 1 5 10 15 66 15 PRT Artificial Sequence BLyS binding polypeptide 66 Thr Lys Phe Cys Asp Arg Leu Thr Met Pro Lys Cys Val Trp Lys 1 5 10 15 67 15 PRT Artificial Sequence BLyS binding polypeptide 67 Asn Thr Phe Cys Pro Asp Pro Leu Thr Gly Arg Cys Val Asn Pro 1 5 10 15 68 15 PRT Artificial Sequence BLyS binding polypeptide 68 Asp Trp Thr Cys Asp Pro Leu Phe His Arg Glu Cys Ile Phe Glu 1 5 10 15 69 15 PRT Artificial Sequence BLyS binding polypeptide 69 Pro Gln Pro Cys Asp Leu Leu Phe Glu Lys Lys Cys Ser Ile Lys 1 5 10 15 70 15 PRT Artificial Sequence BLyS binding polypeptide 70 Arg Trp His Cys Asp Met Leu Ile Asn Pro Ser Cys Leu Pro Asp 1 5 10 15 71 15 PRT Artificial Sequence BLyS binding polypeptide 71 Lys Ile Gln Cys Asp Ile Val Asn Leu Ser Ser Cys Val Tyr Pro 1 5 10 15 72 15 PRT Artificial Sequence BLyS binding polypeptide 72 Leu Asn Ala Cys Asp Ile Val His Pro Asn Tyr Cys Ser Gly Met 1 5 10 15 73 15 PRT Artificial Sequence BLyS binding polypeptide 73 Ala Lys Ala Cys Ser Ile Val Asn Leu Glu Ser Cys Glu Tyr Leu 1 5 10 15 74 15 PRT Artificial Sequence BLyS binding polypeptide 74 Arg Gln Ala Cys Ser Ile Ile Thr Pro Trp Gly Cys Pro Ile Pro 1 5 10 15 75 15 PRT Artificial Sequence BLyS binding polypeptide 75 Ala Asp Asn Cys Thr Val Ala Thr Leu Asp Phe Cys Tyr Trp Thr 1 5 10 15 76 15 PRT Artificial Sequence BLyS binding polypeptide 76 Lys Pro Glu Cys Asn Ile Thr Lys Pro Gln Phe Cys Phe Gly Glu 1 5 10 15 77 15 PRT Artificial Sequence BLyS binding polypeptide 77 Asn Asn Cys Gln Trp Asp Glu Leu Thr Ser Met Cys Asp Pro Phe 1 5 10 15 78 16 PRT Artificial Sequence BLyS binding polypeptide 78 Ser Arg Leu Cys His Met Asp Glu Leu Thr His Val Cys Val His Phe 1 5 10 15 79 16 PRT Artificial Sequence BLyS binding polypeptide 79 Ser Arg Pro Cys Gln Ile Asp Glu Leu Thr Lys Ala Cys Phe Tyr Asn 1 5 10 15 80 16 PRT Artificial Sequence BLyS binding polypeptide 80 Asp Arg Val Cys Lys Leu Asp Phe Leu Thr Tyr Asn Cys Leu Asn His 1 5 10 15 81 16 PRT Artificial Sequence BLyS binding polypeptide 81 His Ser Asn Cys Ile Met Asp Leu Leu Thr Asn Arg Cys Phe Tyr Asp 1 5 10 15 82 16 PRT Artificial Sequence BLyS binding polypeptide 82 Pro Phe Asn Cys Phe His Asp Pro Leu Thr Gly Leu Cys Leu His Ser 1 5 10 15 83 16 PRT Artificial Sequence BLyS binding polypeptide 83 Tyr Asp Ser Cys Thr Tyr Asp Arg Leu Thr Lys Gln Cys Tyr Pro Ser 1 5 10 15 84 16 PRT Artificial Sequence BLyS binding polypeptide 84 Phe His Asp Cys Met Tyr Asp Ala Leu Leu Gly Tyr Cys Leu Pro Tyr 1 5 10 15 85 15 PRT Artificial Sequence BLyS binding polypeptide 85 Asn Arg Ser Cys Asp Pro Leu Thr Arg Pro Lys Ser Cys Gly Leu 1 5 10 15 86 16 PRT Artificial Sequence BLyS binding polypeptide 86 Leu Ser Asn Cys Asp Trp Asp Asp Leu Ile Arg Gln Cys Leu His Asp 1 5 10 15 87 16 PRT Artificial Sequence BLyS binding polypeptide 87 Phe Trp Asp Cys Leu Phe His Pro Asn Ser Arg Tyr Cys Val Leu Ser 1 5 10 15 88 16 PRT Artificial Sequence BLyS binding polypeptide 88 Ser Arg Asp Cys Leu Leu Ser Pro Ala Met Ala Trp Cys Gly Leu Asp 1 5 10 15 89 18 PRT Artificial Sequence BLyS binding polypeptide 89 Gly Gly Asn Cys Tyr Thr Asp Ser Leu Thr Lys Leu His Phe Cys Met 1 5 10 15 Gly Asp 90 16 PRT Artificial Sequence BLyS binding polypeptide 90 Met Cys Pro Arg Asp Pro Leu Thr Lys Ala Lys Leu Cys Asn Trp His 1 5 10 15 91 18 PRT Artificial Sequence BLyS binding polypeptide 91 Pro Asn Gln Cys Gln Asp Asp Leu Thr Lys Gln Trp Tyr Ser Cys His 1 5 10 15 Tyr His 92 18 PRT Artificial Sequence BLyS binding polypeptide 92 Phe Asp Met Cys Phe Asp Ala Leu Thr Lys Gln Asn Phe Tyr Cys Arg 1 5 10 15 Phe His 93 18 PRT Artificial Sequence BLyS binding polypeptide 93 Arg Asn Met Cys Val Asp Arg Leu Thr Lys Leu Gln His Gly Cys Glu 1 5 10 15 Gly Ala 94 18 PRT Artificial Sequence BLyS binding polypeptide 94 Asp Pro Glu Cys Leu Thr Ser Phe Asp Arg Leu Thr Lys Met Cys Trp 1 5 10 15 Pro Trp 95 18 PRT Artificial Sequence BLyS binding polypeptide 95 Asp Asp Glu Cys His Tyr Asp Tyr Leu Thr His Tyr Met Arg Cys Asp 1 5 10 15 Tyr Arg 96 18 PRT Artificial Sequence BLyS binding polypeptide 96 Phe Gly Gly Cys Asn Ile Asp Leu Leu Thr Asn Thr Met Met Cys His 1 5 10 15 Arg Asn 97 18 PRT Artificial Sequence BLyS binding polypeptide 97 His Gly Pro Cys Tyr Trp Asp Glu Leu Thr Met Gln Trp His Cys Asn 1 5 10 15 His His 98 18 PRT Artificial Sequence BLyS binding polypeptide 98 Gly Ala Met Cys Val Asp Leu Leu Thr Tyr Thr Phe Arg Pro Cys Met 1 5 10 15 Tyr Ala 99 18 PRT Artificial Sequence BLyS binding polypeptide 99 Ser Asn Lys Cys Trp Asp Glu Leu Thr His Ala Trp Ala Glu Cys Gly 1 5 10 15 Arg Phe 100 18 PRT Artificial Sequence BLyS binding polypeptide 100 Arg Pro Val Cys Tyr Lys Gly Tyr Asp Ile Leu Thr Thr Gln Cys Met 1 5 10 15 Pro Trp 101 18 PRT Artificial Sequence BLyS binding polypeptide 101 Pro Ser Arg Cys Trp Phe Asp Leu Leu Phe Asn Lys Phe Val Cys Lys 1 5 10 15 Arg Asn 102 18 PRT Artificial Sequence BLyS binding polypeptide 102 Arg Ser Gly Cys Val Tyr Asp Met Leu Leu Met Thr Met Tyr Cys Pro 1 5 10 15 Ser Asn 103 18 PRT Artificial Sequence BLyS binding polypeptide 103 Ser Asn Arg Cys Glu Gly Asp Gln Leu Met Arg Pro Pro Ser Cys Arg 1 5 10 15 His Leu 104 18 PRT Artificial Sequence BLyS binding polypeptide 104 Tyr Arg Met Cys Trp Trp Asp Asp Leu Leu Arg Gly Phe Val Cys Asp 1 5 10 15 Phe His 105 18 PRT Artificial Sequence BLyS binding polypeptide 105 His Asp Gly Cys Tyr Asp Glu Leu Leu Tyr Arg Trp Thr Arg Cys Glu 1 5 10 15 His Arg 106 18 PRT Artificial Sequence BLyS binding polypeptide 106 Trp Ala Trp Cys Phe Asp Glu Leu Val Gln Arg Tyr Phe Thr Cys Phe 1 5 10 15 Asp His 107 18 PRT Artificial Sequence BLyS binding polypeptide 107 Leu Pro Glu Cys Arg Gln Tyr Phe Pro Trp Glu Lys Gln Val Cys Ser 1 5 10 15 Tyr Trp 108 12 PRT Artificial Sequence BLyS binding polypeptide 108 Val His Tyr Asp Ser Leu Thr Lys Met Trp Thr Arg 1 5 10 109 12 PRT Artificial Sequence BLyS binding polypeptide 109 Phe Thr Asp Pro Leu Thr Lys Met Ser Leu His Ser 1 5 10 110 12 PRT Artificial Sequence BLyS binding polypeptide 110 Gly Tyr Asp Val Leu Thr Lys Leu Tyr Phe Val Pro 1 5 10 111 12 PRT Artificial Sequence BLyS binding polypeptide 111 Tyr Tyr Asp Arg Leu Thr Lys Leu Tyr Ser Ser Met 1 5 10 112 12 PRT Artificial Sequence BLyS binding polypeptide 112 Leu Xaa Lys Asp Pro Leu Thr Lys Leu Tyr Ile Ser 1 5 10 113 12 PRT Artificial Sequence BLyS binding polypeptide 113 Gly Tyr Asp Val Leu Thr Lys Leu Xaa Phe Val Pro 1 5 10 114 12 PRT Artificial Sequence BLyS binding polypeptide 114 Arg Leu Tyr Asp Pro Leu Thr Lys Leu Val Leu Ser 1 5 10 115 12 PRT Artificial Sequence BLyS binding polypeptide 115 Met Phe Asp Pro Leu Thr Lys Ile Ala Phe Pro Ala 1 5 10 116 12 PRT Artificial Sequence BLyS binding polypeptide 116 Phe Tyr Asp Ser Leu Thr Lys Thr Asn Leu Arg Asp 1 5 10 117 12 PRT Artificial Sequence BLyS binding polypeptide 117 Gly Ile Tyr Asp Lys Leu Thr Arg Ala Trp Leu Pro 1 5 10 118 12 PRT Artificial Sequence BLyS binding polypeptide 118 Lys Tyr Asp Pro Leu Thr Arg Ala Arg Xaa Pro Leu 1 5 10 119 12 PRT Artificial Sequence BLyS binding polypeptide 119 Tyr Ile Asp Gln Leu Thr Arg Leu Ser Leu Pro Ser 1 5 10 120 12 PRT Artificial Sequence BLyS binding polypeptide 120 His Gln Thr Phe Asp Ile Leu Thr Arg Leu His Phe 1 5 10 121 12 PRT Artificial Sequence BLyS binding polypeptide 121 Trp Gln Phe Asp Val Leu Thr Arg Ser Trp Thr Pro 1 5 10 122 12 PRT Artificial Sequence BLyS binding polypeptide 122 Gly Ala Ala Tyr Asp His Leu Thr Arg Thr Trp Leu 1 5 10 123 12 PRT Artificial Sequence BLyS binding polypeptide 123 Tyr Phe Asp Gln Leu Thr His Leu Ser Ile Lys Lys 1 5 10 124 12 PRT Artificial Sequence BLyS binding polypeptide 124 Ala Trp Asp Pro Leu Thr Met Leu Val Leu Pro Trp 1 5 10 125 12 PRT Artificial Sequence BLyS binding polypeptide 125 Ala Leu Trp Met Asp Pro Leu Thr Gly Leu Ala Phe 1 5 10 126 12 PRT Artificial Sequence BLyS binding polypeptide 126 Trp Gln Phe Asp Val Leu Thr Xaa Ser Trp Thr Pro 1 5 10 127 12 PRT Artificial Sequence BLyS binding polypeptide 127 Trp Thr Asp Pro Leu Thr His Met Glu Ile Tyr His 1 5 10 128 12 PRT Artificial Sequence BLyS binding polypeptide 128 Trp Thr Asp Ser Leu Thr Gly Leu Trp Phe Pro Asp 1 5 10 129 12 PRT Artificial Sequence BLyS binding polypeptide 129 Tyr Thr Asp Pro Leu Thr Gly Ile Val Xaa Pro Phe 1 5 10 130 12 PRT Artificial Sequence BLyS binding polypeptide 130 Tyr Trp Asp Lys Leu Thr Met Leu His Leu Gly Val 1 5 10 131 12 PRT Artificial Sequence BLyS binding polypeptide 131 Tyr Tyr Asp Phe Leu Thr Arg Thr Val Leu Pro Ser 1 5 10 132 12 PRT Artificial Sequence BLyS binding polypeptide 132 Arg Leu Asp Pro Leu Ser Lys Asn Asp Phe Pro Arg 1 5 10 133 12 PRT Artificial Sequence BLyS binding polypeptide 133 Leu Arg Tyr Asp Pro Leu Leu Lys Ser Xaa Ile Tyr 1 5 10 134 12 PRT Artificial Sequence BLyS binding polypeptide 134 Leu Arg Tyr Asp Pro Leu Leu Lys Ser Tyr Ile Tyr 1 5 10 135 12 PRT Artificial Sequence BLyS binding polypeptide 135 Tyr Phe Asp Gln Phe Thr His Leu Ser Ile Lys Lys 1 5 10 136 12 PRT Artificial Sequence BLyS binding polypeptide 136 Tyr Phe Asp Gln Xaa Thr His Leu Ser Ile Lys Lys 1 5 10 137 13 PRT Artificial Sequence BLyS binding polypeptide 137 Glu His Tyr Tyr Thr Asp Pro Leu Thr Gly Ala Arg Ile 1 5 10 138 13 PRT Artificial Sequence BLyS binding polypeptide 138 Glu His Tyr Xaa Thr Asp Pro Leu Thr Gly Ala Arg Ile 1 5 10 139 13 PRT Artificial Sequence BLyS binding polypeptide 139 Glu His Tyr Ser Thr Asp Pro Leu Thr Gly Ala Arg Ile 1 5 10 140 13 PRT Artificial Sequence BLyS binding polypeptide 140 Glu His Tyr Tyr Thr Asp Pro Leu Xaa Gly Xaa Arg Ile 1 5 10 141 13 PRT Artificial Sequence BLyS binding polypeptide 141 Glu His Tyr Tyr Thr Asp Pro Leu Xaa Gly Xaa Arg Xaa 1 5 10 142 13 PRT Artificial Sequence BLyS binding polypeptide 142 Glu His Tyr Tyr Thr Asp Pro Leu Xaa Gly Ala Arg Xaa 1 5 10 143 13 PRT Artificial Sequence BLyS binding polypeptide 143 Glu His Xaa Tyr Thr Asp Pro Leu Asn Gly Ala Arg Xaa 1 5 10 144 13 PRT Artificial Sequence BLyS binding polypeptide 144 Glu His Tyr Tyr Asn Asp Pro Leu Asn Gly Ala Arg Xaa 1 5 10 145 13 PRT Artificial Sequence BLyS binding polypeptide 145 Xaa His Xaa Tyr Asn Asp Pro Leu Asn Gly Ala Arg Xaa 1 5 10 146 13 PRT Artificial Sequence BLyS binding polypeptide 146 Lys Pro Tyr Tyr Asp Pro Ile Thr Lys Met Thr His His 1 5 10 147 13 PRT Artificial Sequence BLyS binding polypeptide 147 Lys Pro Tyr Tyr Asp Pro Ile Thr Lys Met Ser His His 1 5 10 148 13 PRT Artificial Sequence BLyS binding polypeptide 148 Lys Pro Tyr Tyr Asp Pro Ile Ser Lys Met Thr His His 1 5 10 149 13 PRT Artificial Sequence BLyS binding polypeptide 149 Lys Pro Xaa Xaa Asp Pro Ile Ser Lys Met Thr His His 1 5 10 150 13 PRT Artificial Sequence BLyS binding polypeptide 150 Gln Ile Gly Tyr Asp Glu Leu Thr Lys Ala Trp Val Thr 1 5 10 151 13 PRT Artificial Sequence BLyS binding polypeptide 151 Gln Leu Gly Tyr Asp Glu Leu Thr Lys Ala Trp Val Thr 1 5 10 152 13 PRT Artificial Sequence BLyS binding polypeptide 152 Lys Ile Asp Glu Leu Xaa Met Gln Asn Ile Ile Ile Trp 1 5 10 153 13 PRT Artificial Sequence BLyS binding polypeptide 153 Asp His Thr Asp Pro Leu Ile Gln Gly Leu Thr Lys Arg 1 5 10 154 13 PRT Artificial Sequence BLyS binding polypeptide 154 Trp His Asp Pro Leu Lys His Met His Phe His His Glu 1 5 10 155 13 PRT Artificial Sequence BLyS binding polypeptide 155 Lys His Ile Asp Met Glu Thr Gly Leu Ile Leu Gln Asn 1 5 10 156 13 PRT Artificial Sequence BLyS binding polypeptide 156 Met Gln Val Asp Pro Glu Thr Gly Leu Lys Tyr Glu His 1 5 10 157 13 PRT Artificial Sequence BLyS binding polypeptide 157 Xaa Leu Asp Gln His Val Asn Xaa Xaa Xaa Tyr Gln Ser 1 5 10 158 13 PRT Artificial Sequence BLyS binding polypeptide 158 Glu Xaa Xaa Xaa Thr Xaa Xaa Leu Thr Gly Ala Arg Xaa 1 5 10 159 13 PRT Artificial Sequence BLyS binding polypeptide 159 Gly Pro Tyr Asn Ile Xaa Arg Leu Xaa Gly Glu Arg Xaa 1 5 10 160 13 PRT Artificial Sequence BLyS binding polypeptide 160 His Ile Lys Met Leu His Gln Gly Ser Phe Val Gly Val 1 5 10 161 13 PRT Artificial Sequence BLyS binding polypeptide 161 His Pro Thr Asn Thr Xaa Xaa His Gln Xaa Val Tyr Ser 1 5 10 162 13 PRT Artificial Sequence BLyS binding polypeptide 162 His Arg Gly Gln Val Xaa Xaa Leu Asn Gly Met Val Xaa 1 5 10 163 24 PRT Artificial Sequence BLyS binding polypeptide 163 Ala Gly Lys Glu Pro Cys Tyr Phe Tyr Trp Glu Cys Ala Val Ser Gly 1 5 10 15 Pro Gly Pro Glu Gly Gly Gly Lys 20 164 25 PRT Artificial Sequence BLyS binding polypeptide 164 Ala Gly Val Pro Phe Cys Asp Leu Leu Thr Lys His Cys Phe Glu Ala 1 5 10 15 Gly Pro Gly Pro Glu Gly Gly Gly Lys 20 25 165 28 PRT Artificial Sequence BLyS binding polypeptide 165 Gly Ser Ser Arg Leu Cys His Met Asp Glu Leu Thr His Val Cys Val 1 5 10 15 His Phe Ala Pro Pro Gly Pro Glu Gly Gly Gly Lys 20 25 166 29 PRT Artificial Sequence BLyS binding polypeptide 166 Gly Asp Gly Gly Asn Cys Tyr Thr Asp Ser Leu Thr Lys Leu His Phe 1 5 10 15 Cys Met Gly Asp Glu Pro Gly Pro Glu Gly Gly Gly Lys 20 25 167 22 PRT Artificial Sequence BLyS binding polypeptide 167 Gly Tyr Asp Val Leu Thr Lys Leu Tyr Phe Val Pro Gly Gly Pro Gly 1 5 10 15 Pro Glu Gly Gly Gly Lys 20 168 22 PRT Artificial Sequence BLyS binding polypeptide 168 Trp Thr Asp Ser Leu Thr Gly Leu Trp Phe Pro Asp Gly Gly Pro Gly 1 5 10 15 Pro Glu Gly Gly Gly Lys 20 169 24 PRT Artificial Sequence modified BLyS binding polypeptide 169 Ala Gly Lys Glu Pro Cys Tyr Phe Tyr Trp Glu Cys Ala Val Ser Gly 1 5 10 15 Pro Gly Pro Glu Gly Gly Gly Lys 20 170 24 PRT Artificial Sequence modified BLyS binding polypeptide 170 Ala Gly Arg Glu Pro Cys Tyr Phe Tyr Trp Glu Cys Ala Val Ser Gly 1 5 10 15 Pro Gly Pro Glu Gly Gly Gly Lys 20 171 24 PRT Artificial Sequence modified BLyS binding polypeptide 171 Ala Gly Gln Glu Pro Cys Tyr Phe Tyr Trp Glu Cys Ala Val Ser Gly 1 5 10 15 Pro Gly Pro Glu Gly Gly Gly Lys 20 172 25 PRT Artificial Sequence modified BLyS binding polypeptide 172 Ala Gly Asn Xaa Glu Pro Cys Tyr Phe Tyr Trp Glu Cys Ala Val Ser 1 5 10 15 Gly Pro Gly Pro Glu Gly Gly Gly Lys 20 25 173 285 PRT Homo Sapiens 173 Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu 1 5 10 15 Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro 20 25 30 Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu 35 40 45 Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val 50 55 60 Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg 65 70 75 80 Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly 85 90 95 Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu 100 105 110 Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn 115 120 125 Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln 130 135 140 Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys 145 150 155 160 Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser 165 170 175 Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr 180 185 190 Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met 195 200 205 Gly His Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu 210 215 220 Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu 225 230 235 240 Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly 245 250 255 Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu 260 265 270 Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu 275 280 285 174 266 PRT Homo Sapiens 174 Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu 1 5 10 15 Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro 20 25 30 Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu 35 40 45 Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val 50 55 60 Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg 65 70 75 80 Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly 85 90 95 Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu 100 105 110 Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn 115 120 125 Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Gly Ser Tyr 130 135 140 Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu 145 150 155 160 Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile 165 170 175 Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His Leu 180 185 190 Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu Val 195 200 205 Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn Asn 210 215 220 Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu 225 230 235 240 Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp 245 250 255 Val Thr Phe Phe Gly Ala Leu Lys Leu Leu 260 265 175 309 PRT mouse 175 Met Asp Glu Ser Ala Lys Thr Leu Pro Pro Pro Cys Leu Cys Phe Cys 1 5 10 15 Ser Glu Lys Gly Glu Asp Met Lys Val Gly Tyr Asp Pro Ile Thr Pro 20 25 30 Gln Lys Glu Glu Gly Ala Trp Phe Gly Ile Cys Arg Asp Gly Arg Leu 35 40 45 Leu Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Ser Ser Phe Thr Ala 50 55 60 Met Ser Leu Tyr Gln Leu Ala Ala Leu Gln Ala Asp Leu Met Asn Leu 65 70 75 80 Arg Met Glu Leu Gln Ser Tyr Arg Gly Ser Ala Thr Pro Ala Ala Ala 85 90 95 Gly Ala Pro Glu Leu Thr Ala Gly Val Lys Leu Leu Thr Pro Ala Ala 100 105 110 Pro Arg Pro His Asn Ser Ser Arg Gly His Arg Asn Arg Arg Ala Phe 115 120 125 Gln Gly Pro Glu Glu Thr Glu Gln Asp Val Asp Leu Ser Ala Pro Pro 130 135 140 Ala Pro Cys Leu Pro Gly Cys Arg His Ser Gln His Asp Asp Asn Gly 145 150 155 160 Met Asn Leu Arg Asn Ile Ile Gln Asp Cys Leu Gln Leu Ile Ala Asp 165 170 175 Ser Asp Thr Pro Thr Ile Arg Lys Gly Thr Tyr Thr Phe Val Pro Trp 180 185 190 Leu Leu Ser Phe Lys Arg Gly Asn Ala Leu Glu Glu Lys Glu Asn Lys 195 200 205 Ile Val Val Arg Gln Thr Gly Tyr Phe Phe Ile Tyr Ser Gln Val Leu 210 215 220 Tyr Thr Asp Pro Ile Phe Ala Met Gly His Val Ile Gln Arg Lys Lys 225 230 235 240 Val His Val Phe Gly Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys 245 250 255 Ile Gln Asn Met Pro Lys Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala 260 265 270 Gly Ile Ala Arg Leu Glu Glu Gly Asp Glu Ile Gln Leu Ala Ile Pro 275 280 285 Arg Glu Asn Ala Gln Ile Ser Arg Asn Gly Asp Asp Thr Phe Phe Gly 290 295 300 Ala Leu Lys Leu Leu 305 176 290 PRT mouse 176 Met Asp Glu Ser Ala Lys Thr Leu Pro Pro Pro Cys Leu Cys Phe Cys 1 5 10 15 Ser Glu Lys Gly Glu Asp Met Lys Val Gly Tyr Asp Pro Ile Thr Pro 20 25 30 Gln Lys Glu Glu Gly Ala Trp Phe Gly Ile Cys Arg Asp Gly Arg Leu 35 40 45 Leu Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Ser Ser Phe Thr Ala 50 55 60 Met Ser Leu Tyr Gln Leu Ala Ala Leu Gln Ala Asp Leu Met Asn Leu 65 70 75 80 Arg Met Glu Leu Gln Ser Tyr Arg Gly Ser Ala Thr Pro Ala Ala Ala 85 90 95 Gly Ala Pro Glu Leu Thr Ala Gly Val Lys Leu Leu Thr Pro Ala Ala 100 105 110 Pro Arg Pro His Asn Ser Ser Arg Gly His Arg Asn Arg Arg Ala Phe 115 120 125 Gln Gly Pro Glu Glu Thr Glu Gln Asp Val Asp Leu Ser Ala Pro Pro 130 135 140 Ala Pro Cys Leu Pro Gly Cys Arg His Ser Gln His Asp Asp Asn Gly 145 150 155 160 Met Asn Leu Arg Asn Arg Thr Tyr Thr Phe Val Pro Trp Leu Leu Ser 165 170 175 Phe Lys Arg Gly Asn Ala Leu Glu Glu Lys Glu Asn Lys Ile Val Val 180 185 190 Arg Gln Thr Gly Tyr Phe Phe Ile Tyr Ser Gln Val Leu Tyr Thr Asp 195 200 205 Pro Ile Phe Ala Met Gly His Val Ile Gln Arg Lys Lys Val His Val 210 215 220 Phe Gly Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn 225 230 235 240 Met Pro Lys Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala 245 250 255 Arg Leu Glu Glu Gly Asp Glu Ile Gln Leu Ala Ile Pro Arg Glu Asn 260 265 270 Ala Gln Ile Ser Arg Asn Gly Asp Asp Thr Phe Phe Gly Ala Leu Lys 275 280 285 Leu Leu 290 177 239 PRT rat 177 Ala Val Gln Ala Asp Leu Met Ser Leu Arg Met Glu Leu Gln Ser Tyr 1 5 10 15 Arg Ser Ser Ala Thr Pro Ala Ala Pro Gly Ala Pro Gly Leu Ser Ala 20 25 30 Gly Val Lys Leu Pro Thr Pro Ala Ala Pro Gly Pro His Asn Ser Ser 35 40 45 Arg Gly Gln Arg Asn Arg Arg Ala Phe Gln Gly Pro Glu Glu Thr Glu 50 55 60 Gln Asp Val Asp Leu Ser Ala Thr Pro Ala Pro Ser Leu Pro Gly Asn 65 70 75 80 Cys His Ala Ser His His Asp Glu Asn Gly Leu Asn Leu Arg Thr Ile 85 90 95 Ile Gln Asp Cys Leu Gln Leu Ile Ala Asp Ser Asn Thr Pro Thr Ile 100 105 110 Arg Lys Gly Thr Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg 115 120 125 Gly Asn Ala Leu Glu Glu Lys Glu Asn Lys Ile Val Val Arg Gln Thr 130 135 140 Gly Tyr Phe Phe Ile Tyr Ser Gln Val Leu Tyr Thr Asp Pro Ile Phe 145 150 155 160 Ala Met Gly His Val Ile Gln Arg Lys Lys Ile His Val Phe Gly Asp 165 170 175 Glu Leu Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Lys 180 185 190 Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu 195 200 205 Glu Gly Asp Glu Ile Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile 210 215 220 Ser Arg Asn Gly Asp Asp Thr Phe Phe Gly Ala Leu Lys Leu Leu 225 230 235 178 220 PRT rat 178 Ala Val Gln Ala Asp Leu Met Ser Leu Arg Met Glu Leu Gln Ser Tyr 1 5 10 15 Arg Ser Ser Ala Thr Pro Ala Ala Pro Gly Ala Pro Gly Leu Ser Ala 20 25 30 Gly Val Lys Leu Pro Thr Pro Ala Ala Pro Gly Pro His Asn Ser Ser 35 40 45 Arg Gly Gln Arg Asn Arg Arg Ala Phe Gln Gly Pro Glu Glu Thr Glu 50 55 60 Gln Asp Val Asp Leu Ser Ala Thr Pro Val Pro Ser Leu Pro Gly Asn 65 70 75 80 Cys His Ala Ser His His Asp Glu Asn Gly Leu Asn Leu Arg Thr Arg 85 90 95 Thr Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Asn Ala 100 105 110 Leu Glu Glu Lys Glu Asn Lys Ile Val Val Arg Gln Thr Gly Tyr Phe 115 120 125 Phe Ile Tyr Ser Gln Val Leu Tyr Thr Asp Pro Ile Phe Ala Met Gly 130 135 140 His Val Ile Gln Arg Lys Lys Ile His Val Phe Gly Asp Glu Leu Ser 145 150 155 160 Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Lys Thr Leu Pro 165 170 175 Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp 180 185 190 Glu Ile Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Arg Asn 195 200 205 Gly Asp Asp Thr Phe Phe Gly Ala Leu Lys Leu Leu 210 215 220 179 207 PRT rat 179 Ala Val Gln Ala Asp Leu Met Ser Leu Arg Met Glu Leu Gln Ser Tyr 1 5 10 15 Arg Ser Ser Ala Thr Pro Ala Ala Pro Gly Ala Pro Gly Leu Ser Ala 20 25 30 Gly Val Lys Leu Pro Thr Pro Ala Ala Pro Gly Pro His Asn Ser Ser 35 40 45 Arg Gly Gln Arg Asn Arg Arg Ala Phe Gln Gly Pro Glu Glu Thr Val 50 55 60 Ile Gln Asp Cys Leu Gln Leu Ile Ala Asp Ser Asn Thr Pro Thr Ile 65 70 75 80 Arg Lys Gly Thr Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg 85 90 95 Gly Asn Ala Leu Glu Glu Lys Glu Asn Lys Ile Val Val Arg Gln Thr 100 105 110 Gly Tyr Phe Phe Ile Tyr Ser Gln Val Leu Tyr Thr Asp Pro Ile Phe 115 120 125 Ala Met Gly His Val Ile Gln Arg Lys Lys Ile His Val Phe Gly Asp 130 135 140 Glu Leu Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Lys 145 150 155 160 Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu 165 170 175 Glu Gly Asp Glu Val Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile 180 185 190 Ser Arg Asn Gly Asp Asp Thr Phe Phe Gly Ala Leu Lys Leu Leu 195 200 205 180 188 PRT rat 180 Ala Val Gln Ala Asp Leu Met Ser Leu Arg Met Glu Leu Gln Ser Tyr 1 5 10 15 Arg Ser Ser Ala Thr Pro Ala Ala Pro Gly Ala Pro Gly Leu Ser Ala 20 25 30 Gly Val Lys Leu Pro Thr Pro Ala Ala Pro Gly Pro His Asn Ser Ser 35 40 45 Arg Gly Gln Arg Asn Arg Arg Ala Phe Gln Gly Pro Glu Glu Thr Gly 50 55 60 Thr Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Asn Ala 65 70 75 80 Leu Glu Glu Lys Glu Asn Lys Ile Val Val Arg Gln Thr Gly Tyr Phe 85 90 95 Phe Ile Tyr Ser Gln Val Leu Tyr Thr Asp Pro Ile Phe Ala Met Gly 100 105 110 His Val Ile Gln Arg Lys Lys Ile His Val Phe Gly Asp Glu Leu Ser 115 120 125 Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Lys Thr Leu Pro 130 135 140 Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp 145 150 155 160 Glu Ile Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Arg Asn 165 170 175 Gly Asp Asp Thr Phe Phe Gly Ala Leu Lys Leu Leu 180 185 181 243 PRT monkey 181 Lys Asp Arg Lys Leu Leu Ala Ala Ala Leu Leu Leu Ala Leu Leu Ser 1 5 10 15 Cys Cys Leu Met Val Val Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly 20 25 30 Asp Leu Ala Ser Leu Arg Ala Glu Leu Gln Gly His His Ala Glu Lys 35 40 45 Leu Pro Ala Arg Ala Arg Ala Pro Lys Ala Gly Leu Gly Glu Ala Pro 50 55 60 Ala Val Thr Ala Gly Leu Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu 65 70 75 80 Gly Asn Ser Ser Gln Ser Ser Arg Asn Lys Arg Ala Ile Gln Gly Ala 85 90 95 Glu Glu Thr Val Ile Gln Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu 100 105 110 Thr Pro Thr Ile Gln Lys Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu 115 120 125 Ser Phe Lys Arg Gly Ser Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu 130 135 140 Val Lys Glu Thr Gly Tyr Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr 145 150 155 160 Asp Lys Thr Tyr Ala Met Gly His Leu Ile Gln Arg Lys Lys Val His 165 170 175 Val Phe Gly Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys Ile Gln 180 185 190 Asn Met Pro Glu Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile 195 200 205 Ala Lys Leu Glu Glu Gly Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu 210 215 220 Asn Ala Gln Ile Ser Leu Asp Gly Asp Val Thr Phe Phe Gly Ala Leu 225 230 235 240 Lys Leu Leu 182 219 PRT monkey 182 Tyr Gln Val Ala Ala Val Gln Gly Asp Leu Ala Ser Leu Arg Ala Glu 1 5 10 15 Leu Gln Ser His His Ala Glu Lys Leu Pro Ala Arg Ala Arg Ala Pro 20 25 30 Lys Ala Gly Leu Gly Glu Ala Pro Ala Val Thr Ala Gly Leu Lys Ile 35 40 45 Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Ser Ser Arg 50 55 60 Asn Lys Arg Ala Ile Gln Gly Ala Glu Glu Thr Val Ile Gln Asp Cys 65 70 75 80 Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser 85 90 95 Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu 100 105 110 Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe 115 120 125 Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His 130 135 140 Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu 145 150 155 160 Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn 165 170 175 Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu 180 185 190 Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly 195 200 205 Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu 210 215 183 8 PRT Artificial Sequence epitope tag 183 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 184 14 PRT Artificial Sequence concensus BLyS binding polypeptide 184 Ala Asn Trp Tyr Asp Ser Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 185 42 DNA Artificial Sequence coding sequence for BLyS affinity maturation library template 185 gctnnnnnnn nngatnnnct tactnnnctc nnnnnnnnnn nn 42 186 14 PRT Artificial Sequence BLyS binding polypeptide 186 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 187 14 PRT Artificial Sequence BLyS binding polypeptide 187 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Glu 1 5 10 188 14 PRT Artificial Sequence BLyS binding polypeptide 188 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Gly 1 5 10 189 14 PRT Artificial Sequence BLyS binding polypeptide 189 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 190 14 PRT Artificial Sequence BLyS binding polypeptide 190 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Ser Asp 1 5 10 191 14 PRT Artificial Sequence BLyS binding polypeptide 191 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Asn Asp 1 5 10 192 14 PRT Artificial Sequence BLyS binding polypeptide 192 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Thr 1 5 10 193 14 PRT Artificial Sequence BLyS binding polypeptide 193 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 194 14 PRT Artificial Sequence BLyS binding polypeptide 194 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Asn 1 5 10 195 14 PRT Artificial Sequence BLyS binding polypeptide 195 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Val Asp 1 5 10 196 14 PRT Artificial Sequence BLyS binding polypeptide 196 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu His Asp 1 5 10 197 14 PRT Artificial Sequence BLyS binding polypeptide 197 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Thr Asp 1 5 10 198 14 PRT Artificial Sequence BLyS binding polypeptide 198 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro His 1 5 10 199 14 PRT Artificial Sequence BLyS binding polypeptide 199 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Thr Val 1 5 10 200 14 PRT Artificial Sequence BLyS binding polypeptide 200 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Leu Asp 1 5 10 201 14 PRT Artificial Sequence BLyS binding polypeptide 201 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Leu Glu 1 5 10 202 14 PRT Artificial Sequence BLyS binding polypeptide 202 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu His Glu 1 5 10 203 14 PRT Artificial Sequence BLyS binding polypeptide 203 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Arg 1 5 10 204 14 PRT Artificial Sequence BLyS binding polypeptide 204 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Ala Asp 1 5 10 205 14 PRT Artificial Sequence BLyS binding polypeptide 205 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Tyr 1 5 10 206 14 PRT Artificial Sequence BLyS binding polypeptide 206 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Ile 1 5 10 207 14 PRT Artificial Sequence BLyS binding polypeptide 207 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Ile Asp 1 5 10 208 14 PRT Artificial Sequence BLyS binding polypeptide 208 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Arg Asp 1 5 10 209 14 PRT Artificial Sequence BLyS binding polypeptide 209 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 210 14 PRT Artificial Sequence BLyS binding polypeptide 210 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Leu Glu 1 5 10 211 14 PRT Artificial Sequence BLyS binding polypeptide 211 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Arg Val 1 5 10 212 14 PRT Artificial Sequence BLyS binding polypeptide 212 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Glu 1 5 10 213 14 PRT Artificial Sequence BLyS binding polypeptide 213 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 214 14 PRT Artificial Sequence BLyS binding polypeptide 214 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu His Gln 1 5 10 215 14 PRT Artificial Sequence BLyS binding polypeptide 215 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 216 14 PRT Artificial Sequence BLyS binding polypeptide 216 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Arg Val 1 5 10 217 14 PRT Artificial Sequence BLyS binding polypeptide 217 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Gly 1 5 10 218 14 PRT Artificial Sequence BLyS binding polypeptide 218 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Arg Tyr 1 5 10 219 14 PRT Artificial Sequence BLyS binding polypeptide 219 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Tyr 1 5 10 220 14 PRT Artificial Sequence BLyS binding polypeptide 220 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Leu Tyr 1 5 10 221 14 PRT Artificial Sequence BLyS binding polypeptide 221 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Arg Asp 1 5 10 222 14 PRT Artificial Sequence BLyS binding polypeptide 222 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 223 14 PRT Artificial Sequence BLyS binding polypeptide 223 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Leu Gly 1 5 10 224 14 PRT Artificial Sequence BLyS binding polypeptide 224 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Thr His 1 5 10 225 14 PRT Artificial Sequence BLyS binding polypeptide 225 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Thr 1 5 10 226 14 PRT Artificial Sequence BLyS binding polypeptide 226 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Leu Val 1 5 10 227 14 PRT Artificial Sequence BLyS binding polypeptide 227 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Tyr Tyr 1 5 10 228 14 PRT Artificial Sequence BLyS binding polypeptide 228 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Ser Asp 1 5 10 229 14 PRT Artificial Sequence BLyS binding polypeptide 229 Ala Ser Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 230 14 PRT Artificial Sequence BLyS binding polypeptide 230 Ala Ser Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu His Asp 1 5 10 231 14 PRT Artificial Sequence BLyS binding polypeptide 231 Ala Ser Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Gly 1 5 10 232 14 PRT Artificial Sequence BLyS binding polypeptide 232 Ala Ser Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Gln 1 5 10 233 14 PRT Artificial Sequence BLyS binding polypeptide 233 Ala Ser Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Tyr 1 5 10 234 14 PRT Artificial Sequence BLyS binding polypeptide 234 Ala Ser Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro His 1 5 10 235 14 PRT Artificial Sequence BLyS binding polypeptide 235 Ala Ser Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 236 14 PRT Artificial Sequence BLyS binding polypeptide 236 Ala Ser Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Ile 1 5 10 237 14 PRT Artificial Sequence BLyS binding polypeptide 237 Ala Ser Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Glu 1 5 10 238 14 PRT Artificial Sequence BLyS binding polypeptide 238 Ala Phe Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Arg Val 1 5 10 239 14 PRT Artificial Sequence BLyS binding polypeptide 239 Ala Phe Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Glu 1 5 10 240 14 PRT Artificial Sequence BLyS binding polypeptide 240 Ala Phe Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Leu Glu 1 5 10 241 14 PRT Artificial Sequence BLyS binding polypeptide 241 Ala Phe Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 242 14 PRT Artificial Sequence BLyS binding polypeptide 242 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Glu 1 5 10 243 14 PRT Artificial Sequence BLyS binding polypeptide 243 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 244 14 PRT Artificial Sequence BLyS binding polypeptide 244 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu His Asp 1 5 10 245 14 PRT Artificial Sequence BLyS binding polypeptide 245 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Thr Asp 1 5 10 246 14 PRT Artificial Sequence BLyS binding polypeptide 246 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Phe 1 5 10 247 14 PRT Artificial Sequence BLyS binding polypeptide 247 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Leu Asp 1 5 10 248 14 PRT Artificial Sequence BLyS binding polypeptide 248 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Arg 1 5 10 249 14 PRT Artificial Sequence BLyS binding polypeptide 249 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 250 14 PRT Artificial Sequence BLyS binding polypeptide 250 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Thr Ala 1 5 10 251 14 PRT Artificial Sequence BLyS binding polypeptide 251 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Ala Val 1 5 10 252 14 PRT Artificial Sequence BLyS binding polypeptide 252 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Gly 1 5 10 253 14 PRT Artificial Sequence BLyS binding polypeptide 253 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Arg Val 1 5 10 254 14 PRT Artificial Sequence BLyS binding polypeptide 254 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro His 1 5 10 255 14 PRT Artificial Sequence BLyS binding polypeptide 255 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Arg Glu 1 5 10 256 14 PRT Artificial Sequence BLyS binding polypeptide 256 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Ser Asp 1 5 10 257 14 PRT Artificial Sequence BLyS binding polypeptide 257 Ala Thr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 258 14 PRT Artificial Sequence BLyS binding polypeptide 258 Ala Thr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Ala Asp 1 5 10 259 14 PRT Artificial Sequence BLyS binding polypeptide 259 Ala Thr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Thr Ser 1 5 10 260 14 PRT Artificial Sequence BLyS binding polypeptide 260 Ala Thr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Gly 1 5 10 261 14 PRT Artificial Sequence BLyS binding polypeptide 261 Ala Thr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Tyr 1 5 10 262 14 PRT Artificial Sequence BLyS binding polypeptide 262 Ala Thr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Ser Gly 1 5 10 263 14 PRT Artificial Sequence BLyS binding polypeptide 263 Ala Thr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 264 14 PRT Artificial Sequence BLyS binding polypeptide 264 Ala Thr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 265 14 PRT Artificial Sequence BLyS binding polypeptide 265 Ala Asp Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 266 14 PRT Artificial Sequence BLyS binding polypeptide 266 Ala Asp Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Lys 1 5 10 267 14 PRT Artificial Sequence BLyS binding polypeptide 267 Ala Asp Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 268 14 PRT Artificial Sequence BLyS binding polypeptide 268 Ala Asp Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Glu 1 5 10 269 14 PRT Artificial Sequence BLyS binding polypeptide 269 Ala Asp Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu His Gln 1 5 10 270 14 PRT Artificial Sequence BLyS binding polypeptide 270 Ala Glu Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Arg Asp 1 5 10 271 14 PRT Artificial Sequence BLyS binding polypeptide 271 Ala Glu Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 272 14 PRT Artificial Sequence BLyS binding polypeptide 272 Ala Glu Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Tyr 1 5 10 273 14 PRT Artificial Sequence BLyS binding polypeptide 273 Ala Leu Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 274 14 PRT Artificial Sequence BLyS binding polypeptide 274 Ala Leu Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 275 14 PRT Artificial Sequence BLyS binding polypeptide 275 Ala Leu Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Arg Gly 1 5 10 276 14 PRT Artificial Sequence BLyS binding polypeptide 276 Ala Leu Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Leu Gly 1 5 10 277 14 PRT Artificial Sequence BLyS binding polypeptide 277 Ala Met Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 278 14 PRT Artificial Sequence BLyS binding polypeptide 278 Ala Met Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Gln Val 1 5 10 279 14 PRT Artificial Sequence BLyS binding polypeptide 279 Ala Met Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Leu Gly 1 5 10 280 14 PRT Artificial Sequence BLyS binding polypeptide 280 Ala Ala Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 281 14 PRT Artificial Sequence BLyS binding polypeptide 281 Ala Ala Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Ala Asp 1 5 10 282 14 PRT Artificial Sequence BLyS binding polypeptide 282 Ala Ala Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Leu Asp 1 5 10 283 14 PRT Artificial Sequence BLyS binding polypeptide 283 Ala His Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Thr Asp 1 5 10 284 14 PRT Artificial Sequence BLyS binding polypeptide 284 Ala His Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 285 14 PRT Artificial Sequence BLyS binding polypeptide 285 Ala His Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu His Asp 1 5 10 286 14 PRT Artificial Sequence BLyS binding polypeptide 286 Ala His Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 287 14 PRT Artificial Sequence BLyS binding polypeptide 287 Ala Pro Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu His Asp 1 5 10 288 14 PRT Artificial Sequence BLyS binding polypeptide 288 Ala Pro Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 289 14 PRT Artificial Sequence BLyS binding polypeptide 289 Ala Gln Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Glu 1 5 10 290 14 PRT Artificial Sequence BLyS binding polypeptide 290 Ala Gln Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Tyr 1 5 10 291 14 PRT Artificial Sequence BLyS binding polypeptide 291 Ala Gln Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Arg 1 5 10 292 14 PRT Artificial Sequence BLyS binding polypeptide 292 Ala Lys Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 293 14 PRT Artificial Sequence BLyS binding polypeptide 293 Ala Lys Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 294 14 PRT Artificial Sequence BLyS binding polypeptide 294 Ala Lys Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 295 14 PRT Artificial Sequence BLyS binding polypeptide 295 Ala Lys Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Asn Gly 1 5 10 296 14 PRT Artificial Sequence BLyS binding polypeptide 296 Ala Trp Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 297 14 PRT Artificial Sequence BLyS binding polypeptide 297 Ala Val Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Thr Asp 1 5 10 298 14 PRT Artificial Sequence BLyS binding polypeptide 298 Ala Tyr Glu Tyr Asp Pro Leu Thr Lys Leu Trp Leu Leu Tyr 1 5 10 299 14 PRT Artificial Sequence BLyS binding polypeptide 299 Ala Thr Lys Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 300 14 PRT Artificial Sequence BLyS binding polypeptide 300 Ala Thr Leu Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Gly 1 5 10 301 14 PRT Artificial Sequence BLyS binding polypeptide 301 Ala Ile Arg Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Tyr 1 5 10 302 14 PRT Artificial Sequence BLyS binding polypeptide 302 Ala Glu Arg Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro His 1 5 10 303 14 PRT Artificial Sequence BLyS binding polypeptide 303 Ala Asp Arg Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Gln 1 5 10 304 14 PRT Artificial Sequence BLyS binding polypeptide 304 Ala Asn Ser Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Glu 1 5 10 305 14 PRT Artificial Sequence BLyS binding polypeptide 305 Ala Ile Leu Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 306 14 PRT Artificial Sequence BLyS binding polypeptide 306 Ala Asn Trp Phe Asp Pro Leu Thr Lys Leu Trp Leu Pro Gln 1 5 10 307 14 PRT Artificial Sequence BLyS binding polypeptide 307 Ala Asn Trp Phe Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 308 14 PRT Artificial Sequence BLyS binding polypeptide 308 Ala Asn Trp Phe Asp Pro Leu Thr Lys Leu Trp Leu Thr Asp 1 5 10 309 14 PRT Artificial Sequence BLyS binding polypeptide 309 Ala Asn Trp Phe Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 310 14 PRT Artificial Sequence BLyS binding polypeptide 310 Ala Asn Trp Phe Asp Pro Leu Thr Lys Leu Trp Leu Pro Gly 1 5 10 311 14 PRT Artificial Sequence BLyS binding polypeptide 311 Ala Asn Trp Phe Asp Pro Leu Thr Lys Leu Trp Leu Pro Glu 1 5 10 312 14 PRT Artificial Sequence BLyS binding polypeptide 312 Ala Asn Trp Phe Asp Pro Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 313 14 PRT Artificial Sequence BLyS binding polypeptide 313 Ala Asn Trp Phe Asp Pro Leu Thr Lys Leu Trp Leu Pro Asn 1 5 10 314 14 PRT Artificial Sequence BLyS binding polypeptide 314 Ala Asn Trp Phe Asp Pro Leu Thr Lys Leu Trp Leu Ser Glu 1 5 10 315 14 PRT Artificial Sequence BLyS binding polypeptide 315 Ala Asn Trp Phe Asp Pro Leu Thr Lys Leu Trp Leu His Asp 1 5 10 316 14 PRT Artificial Sequence BLyS binding polypeptide 316 Ala Asn Trp Phe Asp Pro Leu Thr Lys Leu Trp Leu Val Asp 1 5 10 317 14 PRT Artificial Sequence BLyS binding polypeptide 317 Ala Tyr Trp Phe Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 318 14 PRT Artificial Sequence BLyS binding polypeptide 318 Ala Tyr Trp Phe Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 319 14 PRT Artificial Sequence BLyS binding polypeptide 319 Ala Tyr Trp Phe Asp Pro Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 320 14 PRT Artificial Sequence BLyS binding polypeptide 320 Ala Gln Trp Phe Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 321 14 PRT Artificial Sequence BLyS binding polypeptide 321 Ala His Trp Phe Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 322 14 PRT Artificial Sequence BLyS binding polypeptide 322 Ala Thr Trp Phe Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 323 14 PRT Artificial Sequence BLyS binding polypeptide 323 Ala Tyr Trp Tyr Asp Ser Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 324 14 PRT Artificial Sequence BLyS binding polypeptide 324 Ala Tyr Trp Tyr Asp Ser Leu Thr Lys Leu Trp Leu His Asp 1 5 10 325 14 PRT Artificial Sequence BLyS binding polypeptide 325 Ala Asn Trp Tyr Asp Ser Leu Thr Lys Leu Trp Ile Pro Asp 1 5 10 326 14 PRT Artificial Sequence BLyS binding polypeptide 326 Ala Asn Trp Tyr Asp Ser Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 327 14 PRT Artificial Sequence BLyS binding polypeptide 327 Ala Asn Trp Tyr Asp Ser Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 328 14 PRT Artificial Sequence BLyS binding polypeptide 328 Ala Asn Trp Tyr Asp Ser Leu Thr Lys Leu Trp Leu Ala Asp 1 5 10 329 14 PRT Artificial Sequence BLyS binding polypeptide 329 Ala Asn Trp Tyr Asp Ser Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 330 14 PRT Artificial Sequence BLyS binding polypeptide 330 Ala Asn Trp Tyr Asp Ser Leu Thr Lys Leu Trp Leu Tyr Glu 1 5 10 331 14 PRT Artificial Sequence BLyS binding polypeptide 331 Ala Gly Trp Tyr Asp Ser Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 332 14 PRT Artificial Sequence BLyS binding polypeptide 332 Ala Val Trp Tyr Asp Ser Leu Thr Lys Leu Trp Leu Thr Asp 1 5 10 333 14 PRT Artificial Sequence BLyS binding polypeptide 333 Ala Asn Trp Tyr Asp Ala Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 334 14 PRT Artificial Sequence BLyS binding polypeptide 334 Ala Tyr Trp Tyr Asp Thr Leu Thr Lys Leu Trp Leu Pro Asn 1 5 10 335 14 PRT Artificial Sequence BLyS binding polypeptide 335 Ala Phe Trp Tyr Asp Pro Leu Thr Asn Leu Trp Leu Leu Glu 1 5 10 336 14 PRT Artificial Sequence BLyS binding polypeptide 336 Ala Tyr Trp Tyr Asp Pro Leu Thr Gly Leu Trp Leu Leu Val 1 5 10 337 14 PRT Artificial Sequence BLyS binding polypeptide 337 Ala Tyr Trp Tyr Asp Pro Leu Thr Gly Leu Trp Leu Leu Tyr 1 5 10 338 14 PRT Artificial Sequence BLyS binding polypeptide 338 Ala Tyr Trp Tyr Asp Pro Leu Thr Gly Leu Trp Leu Arg Val 1 5 10 339 14 PRT Artificial Sequence BLyS binding polypeptide 339 Ala Tyr Trp Tyr Asp Pro Leu Thr Glu Leu Trp Leu Arg Leu 1 5 10 340 14 PRT Artificial Sequence BLyS binding polypeptide 340 Ala Met Trp Tyr Asp Pro Leu Thr Lys Leu Ser Leu Pro Asp 1 5 10 341 14 PRT Artificial Sequence BLyS binding polypeptide 341 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Ser Leu Leu Val 1 5 10 342 14 PRT Artificial Sequence BLyS binding polypeptide 342 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Ser Leu Thr Val 1 5 10 343 14 PRT Artificial Sequence BLyS binding polypeptide 343 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Ser Leu Leu Val 1 5 10 344 14 PRT Artificial Sequence BLyS binding polypeptide 344 Ala Asp Trp Tyr Asp Pro Leu Thr Lys Leu Ser Leu Leu Leu 1 5 10 345 14 PRT Artificial Sequence BLyS binding polypeptide 345 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Arg Leu Leu Glu 1 5 10 346 14 PRT Artificial Sequence BLyS binding polypeptide 346 Ala Asp Trp Tyr Asp Pro Leu Thr Lys Leu Arg Leu Leu Val 1 5 10 347 14 PRT Artificial Sequence BLyS binding polypeptide 347 Ala Asp Trp Tyr Asp Pro Leu Thr Lys Leu Arg Leu Ile Val 1 5 10 348 14 PRT Artificial Sequence BLyS binding polypeptide 348 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Tyr Leu Pro Asp 1 5 10 349 14 PRT Artificial Sequence BLyS binding polypeptide 349 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Gly Leu Leu Val 1 5 10 350 14 PRT Artificial Sequence BLyS binding polypeptide 350 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Thr Leu Leu Val 1 5 10 351 14 PRT Artificial Sequence BLyS binding polypeptide 351 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Leu Leu Pro Asn 1 5 10 352 14 PRT Artificial Sequence BLyS binding polypeptide 352 Ala Ser Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro Asp 1 5 10 353 14 PRT Artificial Sequence BLyS binding polypeptide 353 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro Asp 1 5 10 354 14 PRT Artificial Sequence BLyS binding polypeptide 354 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Ser Asp 1 5 10 355 14 PRT Artificial Sequence BLyS binding polypeptide 355 Ala Ser Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro Val 1 5 10 356 14 PRT Artificial Sequence BLyS binding polypeptide 356 Ala Asp Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro Val 1 5 10 357 14 PRT Artificial Sequence BLyS binding polypeptide 357 Ala Ser Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro Lys 1 5 10 358 14 PRT Artificial Sequence BLyS binding polypeptide 358 Ala Lys Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro Asp 1 5 10 359 14 PRT Artificial Sequence BLyS binding polypeptide 359 Ala Ser Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Leu Glu 1 5 10 360 14 PRT Artificial Sequence BLyS binding polypeptide 360 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro Ala 1 5 10 361 14 PRT Artificial Sequence BLyS binding polypeptide 361 Ala Thr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro Asp 1 5 10 362 14 PRT Artificial Sequence BLyS binding polypeptide 362 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro Glu 1 5 10 363 14 PRT Artificial Sequence BLyS binding polypeptide 363 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro Asp 1 5 10 364 14 PRT Artificial Sequence BLyS binding polypeptide 364 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro Gly 1 5 10 365 14 PRT Artificial Sequence BLyS binding polypeptide 365 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro His 1 5 10 366 14 PRT Artificial Sequence BLyS binding polypeptide 366 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro Val 1 5 10 367 14 PRT Artificial Sequence BLyS binding polypeptide 367 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro Asp 1 5 10 368 14 PRT Artificial Sequence BLyS binding polypeptide 368 Ala Gly Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro Asp 1 5 10 369 14 PRT Artificial Sequence BLyS binding polypeptide 369 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro Thr 1 5 10 370 14 PRT Artificial Sequence BLyS binding polypeptide 370 Ala Lys Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro Ala 1 5 10 371 14 PRT Artificial Sequence BLyS binding polypeptide 371 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Phe Asp 1 5 10 372 14 PRT Artificial Sequence BLyS binding polypeptide 372 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Ala Asp 1 5 10 373 14 PRT Artificial Sequence BLyS binding polypeptide 373 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro Tyr 1 5 10 374 14 PRT Artificial Sequence BLyS binding polypeptide 374 Ala Asp Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Arg Asp 1 5 10 375 14 PRT Artificial Sequence BLyS binding polypeptide 375 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Val Pro Asp 1 5 10 376 14 PRT Artificial Sequence BLyS binding polypeptide 376 Ala Asp Trp Tyr Asp Pro Leu Thr Lys Leu Trp Val Pro Ala 1 5 10 377 14 PRT Artificial Sequence BLyS binding polypeptide 377 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Val Pro Asn 1 5 10 378 14 PRT Artificial Sequence BLyS binding polypeptide 378 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Val Pro Glu 1 5 10 379 14 PRT Artificial Sequence BLyS binding polypeptide 379 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Val Pro Gln 1 5 10 380 14 PRT Artificial Sequence BLyS binding polypeptide 380 Ala Glu Trp Tyr Asp Pro Leu Thr Lys Leu Trp Val Pro Lys 1 5 10 381 14 PRT Artificial Sequence BLyS binding polypeptide 381 Ala Gln Trp Tyr Asp Pro Leu Thr Lys Leu Trp Val Pro Val 1 5 10 382 14 PRT Artificial Sequence BLyS binding polypeptide 382 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Val Pro Tyr 1 5 10 383 14 PRT Artificial Sequence BLyS binding polypeptide 383 Ala Leu Trp Tyr Asp Pro Leu Thr Lys Leu Trp Val Pro Tyr 1 5 10 384 14 PRT Artificial Sequence BLyS binding polypeptide 384 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Val Pro Gly 1 5 10 385 14 PRT Artificial Sequence BLyS binding polypeptide 385 Ala Ser Trp Tyr Asp Pro Leu Thr Lys Leu Trp Ile Pro Tyr 1 5 10 386 14 PRT Artificial Sequence BLyS binding polypeptide 386 Ala Asp Trp Tyr Asp Pro Leu Thr Lys Leu Trp Ile Pro Gly 1 5 10 387 14 PRT Artificial Sequence BLyS binding polypeptide 387 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Ile Pro Tyr 1 5 10 388 14 PRT Artificial Sequence BLyS binding polypeptide 388 Ala Lys Trp Tyr Asp Pro Leu Thr Lys Leu Trp Ile Pro Tyr 1 5 10 389 14 PRT Artificial Sequence BLyS binding polypeptide 389 Ala Ile Trp Tyr Asp Pro Leu Thr Lys Leu Trp Ile Pro Asn 1 5 10 390 14 PRT Artificial Sequence BLyS binding polypeptide 390 Ala Thr Trp Tyr Asp Pro Leu Thr Lys Leu Trp Ile Pro Gln 1 5 10 391 14 PRT Artificial Sequence BLyS binding polypeptide 391 Ala Ser Trp Tyr Asp Pro Leu Thr Asn Leu Trp Val Pro Asp 1 5 10 392 14 PRT Artificial Sequence BLyS binding polypeptide 392 Ala Tyr Glu Tyr Asp Pro Leu Thr Asn Leu Trp Leu Leu Tyr 1 5 10 393 14 PRT Artificial Sequence BLyS binding polypeptide 393 Ala Tyr Trp Tyr Asp Pro Leu Thr Asn Leu Ser Leu Leu Val 1 5 10 394 14 PRT Artificial Sequence BLyS binding polypeptide 394 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Ser Ile Leu Glu 1 5 10 395 14 PRT Artificial Sequence BLyS binding polypeptide 395 Ala Asn Trp Tyr Asp Ser Leu Thr Lys Leu Trp Ile Pro Tyr 1 5 10 396 14 PRT Artificial Sequence BLyS binding polypeptide 396 Ala His Trp Phe Asp Pro Leu Thr Gln Leu Lys Ile Arg Val 1 5 10 397 14 PRT Artificial Sequence BLyS binding polypeptide 397 Ala Tyr Trp Cys Asp Pro Leu Thr Lys Leu Cys Ile Leu Glu 1 5 10 398 14 PRT Artificial Sequence BLyS binding polypeptide 398 Ala Asn Ser Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro Tyr 1 5 10 399 14 PRT Artificial Sequence BLyS binding polypeptide 399 Ala Asn Leu Tyr Asp Pro Leu Thr Lys Leu Trp Val Pro Tyr 1 5 10 400 14 PRT Artificial Sequence BLyS binding polypeptide 400 Ala Asn Trp Tyr Asp Ala Leu Thr Lys Leu Trp Leu His Asp 1 5 10 401 14 PRT Artificial Sequence BLyS binding polypeptide 401 Ala Asn Trp Tyr Asp Ser Leu Thr Lys Leu Trp Phe Pro Asp 1 5 10 402 14 PRT Artificial Sequence BLyS binding polypeptide 402 Ala Thr Ser Tyr Asp Ser Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 403 14 PRT Artificial Sequence BLyS binding polypeptide 403 Ala Cys Trp Tyr Asp Ser Leu Thr Lys Leu Cys His Arg Glu 1 5 10 404 14 PRT Artificial Sequence BLyS binding polypeptide 404 Ala Ile Gly Asn Asp Pro Leu Thr Lys Leu Trp Ile Pro Tyr 1 5 10 405 14 PRT Artificial Sequence BLyS binding polypeptide 405 Ala Asn Trp Gln Asp Cys Leu Thr Lys Leu Cys Leu Ala Gly 1 5 10 406 14 PRT Artificial Sequence BLyS binding polypeptide 406 Ala Tyr Trp Phe Asp Pro Leu Thr Asn Leu Trp Leu Leu Glu 1 5 10 407 14 PRT Artificial Sequence BLyS binding polypeptide 407 Ala Tyr Trp Tyr Asp Pro Leu Thr Asn Leu Ser Leu Leu Val 1 5 10 408 14 PRT Artificial Sequence BLyS binding polypeptide 408 Ala Asn Cys Phe Asp Ser Leu Thr Arg Leu Trp Leu Cys Asp 1 5 10 409 14 PRT Artificial Sequence BLyS binding polypeptide 409 Ala Cys Ala Tyr Asp Ala Leu Thr Lys Leu Cys Leu Pro Ala 1 5 10 410 14 PRT Artificial Sequence BLyS binding polypeptide 410 Ala Asn Trp Tyr Asp Pro Leu Thr Asn Leu Ser Leu Leu Leu 1 5 10 411 14 PRT Artificial Sequence BLyS binding polypeptide 411 Ala Tyr Trp Tyr Asp Pro Leu Thr Gln Leu Ser Leu Leu Val 1 5 10 412 14 PRT Artificial Sequence BLyS binding polypeptide 412 Ala Tyr Arg Tyr Asp Ala Leu Thr Gly Leu Trp Leu Leu Tyr 1 5 10 413 14 PRT Artificial Sequence BLyS binding polypeptide 413 Ala Tyr Trp Asn Asp Pro Leu Thr Lys Leu Lys Leu Arg Leu 1 5 10 414 14 PRT Artificial Sequence BLyS binding polypeptide 414 Ala Tyr Trp Tyr Asp Pro Leu Thr Gln Leu Ser Leu Leu Val 1 5 10 415 14 PRT Artificial Sequence BLyS binding polypeptide 415 Ala Tyr Arg Tyr Asp Ala Leu Thr Gly Leu Trp Leu Leu Tyr 1 5 10 416 14 PRT Artificial Sequence BLyS binding polypeptide 416 Ala Tyr Arg Tyr Asp Ser Leu Thr Asn Leu Trp Leu Leu Tyr 1 5 10 417 14 PRT Artificial Sequence BLyS binding polypeptide 417 Ala Tyr Trp Tyr Asp Pro Leu Thr Lys Leu Ser Ile Leu Glu 1 5 10 418 14 PRT Artificial Sequence BLyS binding polypeptide 418 Ala Ser Cys Tyr Asp Pro Leu Thr Lys Leu Cys Phe Pro Val 1 5 10 419 14 PRT Artificial Sequence BLyS binding polypeptide 419 Ala Phe Trp Phe Asp Pro Leu Thr Gly Leu Trp Leu Leu Glu 1 5 10 420 14 PRT Artificial Sequence BLyS binding polypeptide 420 Ala His Trp Tyr Asp Pro Leu Thr Lys Leu Ser Ile Arg Val 1 5 10 421 14 PRT Artificial Sequence BLyS binding polypeptide 421 Ala Pro Trp Tyr Asp Ser Leu Thr Lys Leu Trp Phe Pro Ser 1 5 10 422 14 PRT Artificial Sequence BLyS binding polypeptide 422 Ala Asn Cys Tyr Asp Thr Leu Thr Lys Leu Trp Leu Thr Cys 1 5 10 423 14 PRT Artificial Sequence BLyS binding polypeptide 423 Ala Asn Trp Tyr Asp Ser Leu Thr Lys Leu Ser Leu Pro Asp 1 5 10 424 14 PRT Artificial Sequence BLyS binding polypeptide 424 Ala Tyr Ala Tyr Asp Phe Leu Thr Gln Leu Ser Leu Pro Asp 1 5 10 425 14 PRT Artificial Sequence BLyS binding polypeptide 425 Ala Phe Arg Tyr Asp Ser Leu Thr Gly Leu Trp Leu Arg Tyr 1 5 10 426 14 PRT Artificial Sequence BLyS binding polypeptide 426 Ala Asn Cys Tyr Asp Ser Leu Thr Lys Leu Trp Leu Pro Cys 1 5 10 427 14 PRT Artificial Sequence BLyS binding polypeptide 427 Ala Asn Gly Tyr Asp Leu Leu Thr Asn Leu Ser Val Ser Asp 1 5 10 428 14 PRT Artificial Sequence BLyS binding polypeptide 428 Ala Asn Trp Tyr Asp Pro Leu Thr Arg Leu Trp Ile Pro Val 1 5 10 429 14 PRT Artificial Sequence BLyS binding polypeptide 429 Ala Leu Lys Phe Asp Tyr Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 430 14 PRT Artificial Sequence BLyS binding polypeptide 430 Ala Tyr Arg Tyr Asp Ser Leu Thr Lys Leu Trp Leu Pro Gly 1 5 10 431 14 PRT Artificial Sequence BLyS binding polypeptide 431 Ala Tyr Cys Tyr Asp Ser Leu Thr Lys Leu Trp Ile Pro Asp 1 5 10 432 14 PRT Artificial Sequence BLyS binding polypeptide 432 Ala Ser Trp Glu Asp Ser Leu Thr Lys Leu Trp Leu Ser Lys 1 5 10 433 14 PRT Artificial Sequence BLyS binding polypeptide 433 Ala Tyr Trp Tyr Asp Ser Leu Thr Gly Leu Ser Leu Leu Val 1 5 10 434 14 PRT Artificial Sequence BLyS binding polypeptide 434 Ala Tyr Trp Tyr Asp Pro Leu Thr Tyr Leu Arg Leu Arg Val 1 5 10 435 14 PRT Artificial Sequence BLyS binding polypeptide 435 Ala Lys Cys Tyr Asp Ser Leu Thr Asn Leu Trp Leu Cys Asp 1 5 10 436 10 PRT Artificial Sequence core peptide of high affinity BLyS binders 436 Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu 1 5 10 437 17 PRT Artificial Sequence BLyS binding polypeptide 437 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp Gly Gly 1 5 10 15 Lys 438 15 PRT Artificial Sequence BLyS binding polypeptide 438 Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp Gly Gly Lys 1 5 10 15 439 13 PRT Artificial Sequence BLyS binding polypeptide 439 Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Gly Gly Lys 1 5 10 440 17 PRT Artificial Sequence BLyS binding polypeptide 440 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Val Gly Gly 1 5 10 15 Lys 441 17 PRT Artificial Sequence BLyS binding polypeptide 441 Ala Asn Trp Phe Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp Gly Gly 1 5 10 15 Lys 442 17 PRT Artificial Sequence BLyS binding polypeptide 442 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Ser Leu Pro Asp Gly Gly 1 5 10 15 Lys 443 17 PRT Artificial Sequence BLyS binding polypeptide 443 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro Asp Gly Gly 1 5 10 15 Lys 444 17 PRT Artificial Sequence BLyS binding polypeptide 444 Ala Asn Trp Tyr Asp Ser Leu Thr Lys Leu Trp Leu Pro Asp Gly Gly 1 5 10 15 Lys 445 585 PRT HomoSapiens 445 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 446 4 PRT Artificial Sequence recurring structural motif of BLyS binding polypeptides 446 Asp Xaa Leu Thr 1 447 14 PRT Artificial Sequence BLyS binding polypeptide 447 Ala Xaa Xaa Xaa Asp Xaa Leu Thr Xaa Leu Xaa Xaa Xaa Xaa 1 5 10 448 10 PRT Artificial Sequence BLyS binding polypeptide 448 Xaa Xaa Asp Xaa Leu Thr Xaa Leu Xaa Xaa 1 5 10 449 14 PRT Artificial Sequence BLyS affinity maturation library template 449 Ala Xaa Xaa Xaa Asp Xaa Leu Thr Xaa Leu Xaa Xaa Xaa Xaa 1 5 10 450 16 PRT Artificial Sequence BLyS binding polypeptide 450 Ala Gly Lys Glu Pro Cys Tyr Phe Tyr Trp Glu Cys Ala Val Ser Gly 1 5 10 15 451 17 PRT Artificial Sequence BLyS binding polypeptide 451 Ala Gly Val Pro Phe Cys Asp Leu Leu Thr Lys His Cys Phe Glu Ala 1 5 10 15 Gly 452 20 PRT Artificial Sequence BLyS binding polypeptide 452 Gly Ser Ser Arg Leu Cys His Met Asp Glu Leu Thr His Val Cys Val 1 5 10 15 His Phe Ala Pro 20 453 21 PRT Artificial Sequence BLyS binding polypeptide 453 Gly Asp Gly Gly Asn Cys Tyr Thr Asp Ser Leu Thr Lys Leu His Phe 1 5 10 15 Cys Met Gly Asp Glu 20 454 14 PRT Artificial Sequence BLyS binding polypeptide 454 Gly Tyr Asp Val Leu Thr Lys Leu Tyr Phe Val Pro Gly Gly 1 5 10 455 14 PRT Artificial Sequence BLyS binding polypeptide 455 Trp Thr Asp Ser Leu Thr Gly Leu Trp Phe Pro Asp Gly Gly 1 5 10 456 12 PRT Artificial Sequence BLyS binding polypeptide 456 Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 457 10 PRT Artificial Sequence BLyS binding polypeptide 457 Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu 1 5 10 458 14 PRT Artificial Sequence BLyS binding polypeptide 458 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Ser Leu Pro Asp 1 5 10 

1. A B Lymphocyte Stimulator (BLyS) binding polypeptide comprising the amino acid sequence: Asp-Xaa-Leu-Thr (SEQ ID NO: 446), wherein Xaa is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala.
 2. The polypeptide according to claim 1, wherein Xaa is Pro or Ser.
 3. The polypeptide according to claim 1, wherein said polypeptides comprises the amino acid sequence: X₁-X₂-Asp-X₄-Leu-Thr-X₇-Leu-X₉-X₁₀ (SEQ ID NO: 448), wherein X₁ is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly, or Ser; X₂ is Tyr, Phe, Glu, Cys, Asn; X₄ is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala; X₇ is Lys, Asn, Gln, Gly, or Arg; X₉ is Trp, Ser, Thr, Arg, Cys, Tyr, or Lys; and X₁₀ is Leu, Phe, Val, Ile, or His.
 4. The polypeptide according to claim 3, wherein said polypeptide comprises the amino acid sequence: Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu (SEQ ID NO: 436).
 5. The polypeptide according to claim 3, wherein said polypeptide comprises the amino acid sequence: Ala-X₂-X₃-X₄-Asp-X₆-Leu-Thr-X₉-Leu-X₁₁-X₁₂-X₁₃-X₁₄ (SEQ ID NO: 447), wherein X₂ is any amino acid except Arg; X₃ is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly, or Ser; X₄ is Tyr, Phe, Glu, Cys, Asn; X₆ is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala; X₉ is Lys, Asn, Gln, Gly, or Arg; X₁₁ is Trp, Ser, Thr, Arg, Cys, Tyr, or Lys; X₁₂ is Leu, Phe, Val, Ile, or His; X₁₃ is Pro, Leu, His, Ser, Arg, Asn, Gln, Thr, Val, Ala, Cys, Ile, Phe, or Tyr; and X₁₄ is Asp, Glu, Asn, Val, His, Gln, Arg, Gly, Ser, Tyr, Ala, Cys, Lys, Ile, Thr or Leu.
 6. The polypeptide according to claim 3, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 186-435 as depicted in Table
 14. 7. The polypeptide according to claim 3, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 437-444 as depicted in Table
 15. 8. The polypeptide according to claim 1, comprising an amino acid sequence selected from the group consisting of: Ala-Gly-Lys-Glu-Pro-Cys-Tyr-Phe-Tyr-Trp-Glu-Cys-Ala-Val-Ser-Gly; (SEQ ID NO:450) Ala-Gly-Val-Pro-Phe-Cys-Asp-Leu-Leu-Thr-Lys-His-Cys-Phe-Glu-Ala-Gly; (SEQ ID NO:451) Gly-Ser-Ser-Arg-Leu-Cys-His-Met-Asp-Glu-Leu-Thr-His-Val-Cys-Val-His-Phe-Ala-Pro; (SEQ ID NO:452) Gly-Asp-Gly-Gly-Asn-Cys-Tyr-Thr-Asp-Ser-Leu-Thr-Lys-Leu-His-Phe-Cys-Met-Gly-Asp-Glu; (SEQ ID NO:453) Gly-Tyr-Asp-Val-Leu-Thr-Lys-Leu-Tyr-Phe-Val-Pro-Gly-Gly; (SEQ ID NO:454) Trp-Thr-Asp-Ser-Leu-Thr-Gly-Leu-Trp-Phe-Pro-Asp-Gly-Gly; (SEQ ID NO:455) Ala-Asn-Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu-Pro-Asp; (SEQ ID NO:186) Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu-Pro-Asp; (SEQ ID NO:456) Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu; (SEQ ID NO:457) Ala-Asn-Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu-Pro-Val; (SEQ ID NO:189) Ala-Asn-Trp-Phe-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu-Pro-Asp; (SEQ ID NO:309) Ala-Asn-Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Ser-Leu-Pro-Asp; (SEQ ID NO:458) Ala-Asn-Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Phe-Pro-Asp; (SEQ ID NO:353) Ala-Asn-Trp-Tyr-Asp-Ser-Leu-Thr-Lys-Leu-Trp-Leu-Pro-Asp. (SEQ ID NO:327)


9. A BLyS binding polypeptide comprising an amino acid sequence according to one of the following formulae: (H) Cys-X₂-Phe-X₄-Trp-Glu-Cys (SEQ ID NO: 8), wherein X₂ is Phe, Trp, or Tyr; and X₄ is Pro or Tyr; or (I) Cys-X₂-X₃-X₄-X₅-X₆-X₇-Cys (SEQ ID NO: 9), wherein X₂ is Asp, Ile, Leu, or Tyr; X₃ is Arg, Asp, Glu, His, Ile, Leu, Lys, Phe, Pro, Tyr, or Val; X₄ is His, Leu, Lys, or Phe; X₅ is Leu, Pro, or Thr; X₆ is Arg, Asn, Gly, His, Ile, Lys, Met, or Trp; and X₇ is Ala, Asn, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Ser, Trp, Tyr, or Val; or (J) Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-Cys (SEQ ID NO: 10), wherein X₂ is Asn, Asp, Pro, Ser, or Thr; X₃ is Arg, Asp, Ile, Leu, Met, Pro, or Val; X₄ is Ala, Ile, Leu, Pro, Thr, or Val; X₅ is Asn, His, Ile, Leu, Lys, Phe, or Thr; X₆ is Asn, Glu, Gly, His, Leu, Lys, Met, Pro, or Thr; X₇ is Arg, Asn, Asp, Gln, Glu, Gly, Ile, Lys, Met, Pro, Ser, or Trp; X₈ is Arg, Glu, Gly, Lys, Phe, Ser, Trp, or Tyr; or (K) Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-Cys (SEQ ID NO: 11), wherein X₂ is Asp, Gln, His, Ile, Leu, Lys, Met, Phe, or Thr; X₃ is His, Ile, Leu, Met, Phe, Pro, Trp, or Tyr; X₄ is Asp, His, Leu, or Ser; X₅ is Ala, Arg, Asp, Glu, Leu, Phe, Pro, or Thr; X₆ is Ala, Arg, Asn, or Leu; X₇ is Ile, Leu, Met, Pro, Ser, or Thr; X₈ is Ala, Arg, Asn, Gly, His, Lys, Ser, or Tyr; X₉ is Ala, Arg, Asn, Gln, Leu, Met, Ser, Trp, Tyr, or Val; or (L) Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys (SEQ ID NO: 12), wherein X₂ is Arg, Asn, Gln, Glu, His, Leu, Phe, Pro, Trp, Tyr, or Val; X₃ is Arg, Asp, Gln, Gly, Ile, Lys, Phe, Thr, Trp or Tyr; X₄ is Ala, Arg, Asp, Glu, Gly, Leu, Ser, or Tyr; X₅ is Asp, Gln, Glu, Leu, Met, Phe, Pro, Ser, or Tyr; X₆ is Asp, Leu, Pro, Thr, or Val; X₇ is Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp or Tyr; X₈ is Ala, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, or Thr; X₉ is Ala, Asn, Gln, Gly, Leu, Lys, Phe, Pro, Thr, Trp, or Tyr; X₁₀ is Ala, Arg, Gln, His, Lys, Met, Phe, Pro, Thr, Trp, or Tyr; and X₁₁ is Arg, Gln, Glu, Gly, His, Leu, Met, Phe, Pro, Ser, Thr, Tyr, or Val.
 10. The polypeptide according to claim 9, wherein (a) said polypeptide comprises an amino acid sequence of the formula: Cys-X₂-Phe-X₄-Trp-Glu-Cys (SEQ ID NO: 8), and the following amino acid positions are independently selected as follows: X₂ is Tyr; X₄ is Pro; or combinations of such selections; or (b) said polypeptide comprises an amino acid sequence of the following formula: Cys-X₂-X₃-X₄-X₅-X₆-X₇-Cys (SEQ ID NO: 9), and the following amino acid positions are independently selected as follows: X₂ is Asp or Leu; X₃ is Glu or Leu; X₄ is His or Leu; X₅ is Thr or Pro; X₆ is Lys; or combinations of such selections; or (c) said polypeptide comprises an amino acid sequence of the following formula: Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-Cys (SEQ ID NO: 10), and the following amino acid positions are independently selected as follows: X₂ is Asp; X₃ is Ile; X₄ is Val or Leu; X₅ is Thr; X₆ is Leu; X₈ is Ser; or combinations of such selections; or (d) said polypeptide comprises an amino acid sequence of the following formula: Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-Cys (SEQ ID NO: 11), and the following amino acid positions are independently selected as follows: X₄ is Asp; X₅ is Glu or Pro; X₆ is Leu; X₇ is Thr; or combinations of such selections; or (e) said polypeptide comprises an amino acid sequence of the following formula: Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys (SEQ ID NO: 12), and the following amino acid positions are independently selected as follows: X₂ is Trp, Tyr, or Val; X₃ is Asp; X₄ is Asp; X₅ is Leu; X₆ is Leu or Thr; X₇ is Lys or Thr; X₈ is Arg or Leu; X₉ is Thr or Trp; X₁₀ is Met or Phe; X₁₁ is Val; or combinations of such selections.
 11. A BLyS binding polypeptide comprising an amino acid sequence of the following formula: (A) X₁-X₂-X₃-Cys-X₅-Phe-X₇-Trp-Glu-Cys-X₁₁-X₁₂-X₁₃ (SEQ ID NO: 1), wherein X₁ is Ala, Asn, Lys, or Ser; X₂ is Ala, Glu, Met, Ser, or Val; X₃ is Ala, Asn, Lys, or Pro; X₅ is Phe, Trp, or Tyr; X₇ is Pro or Tyr; X₁₁ is Ala, Gln, His, Phe, or Val; X₁₂ is Asn, Gln, Gly, His, Ser, or Val; and X₁₃ is Ala, Asn, Gly, Ile, Pro, or Ser; or (B) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-Cys-X₁₂-X₁₃-X₁₄ (SEQ ID NO: 2), wherein X₁ is Ala, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, or is absent; X₂ is Ala, Asn, Asp, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X₃ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val; X₅ is Asp, Ile, Leu, or Tyr; X₆ is Arg, Asp, Glu, His, Ile, Leu, Lys, Phe, Pro, Tyr, or Val; X₇ is His, Leu, Lys, or Phe; X₈ is Leu, Pro, or Thr; X₉ is Arg, Asn, Gly, His, Ile, Lys, Met, or Trp; X₁₀ is Ala, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Ser, Trp, Tyr, or Val; X₁₂ is Asp, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Ser, Trp, Tyr, or Val; X₁₃ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and X₁₄ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Trp, Tyr, Val, or is absent; or (C) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys-X₁₃-X₁₄-X₁₅ (SEQ ID NO: 3), wherein X₁ is Ala, Arg, Asn, Asp, Leu, Lys, Phe, Pro, Ser, or Thr; X₂ is Asn, Asp, Gln, His, Ile, Lys, Pro, Thr, or Trp; X₃ is Ala, Arg, Asn, Gln, Glu, His, Phe, Pro, or Thr; X₅ is Asn, Asp, Pro, Ser, or Thr; X₆ is Arg, Asp, Ile, Leu, Met, Pro, or Val; X₇ is Ala, Ile, Leu, Pro, Thr, or Val; X₈ is Asn, His, Ile, Leu, Lys, Phe, or Thr; X₉ is Asn, Glu, Gly, His, Leu, Lys, Met, Pro, or Thr; X₁₀ is Arg, Asn, Asp, Gln, Glu, Gly, Ile, Lys, Met, Pro, Ser, or Trp; X₁₁ is Arg, Glu, Gly, Lys, Phe, Ser, Trp, or Tyr; X₁₃ is Gln, Glu, Ile, Leu, Phe, Pro, Ser, Tyr, or Val; X₁₄ is Asn, Gly, Ile, Phe, Pro, Thr, Trp, or Tyr; and X₁₅ is Asn, Asp, Glu, Leu, Lys, Met, Pro, or Thr; or (D) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-Cys-X₁₄-X₁₅-X₁₆ (SEQ ID NO: 4), wherein X₁ is Asn, Asp, His, Leu, Phe, Pro, Ser, Tyr, or is absent; X₂ is Arg, Asn, Asp, His, Phe, Ser, or Trp; X₃ is Asn, Asp, Leu, Pro, Ser, or Val; X₅ is Asp, Gln, His, Ile, Leu, Lys, Met, Phe, or Thr; X₆ is His, Ile, Leu, Met, Phe, Pro, Trp, or Tyr; X₇ is Asp, His, Leu, or Ser; X₈ is Ala, Arg, Asp, Glu, Leu, Phe, Pro, or Thr; X₉ is Ala, Arg, Asn, or Leu; X₁₀ is Ile, Leu, Met, Pro, Ser, or Thr; X₁₁ is Ala, Arg, Asn, Gly, His, Lys, Ser, or Tyr; X₁₂ is Ala, Arg, Asn, Gln, Leu, Met, Ser, Trp, Tyr, or Val; X₁₄ is Asp, Gly, Leu, Phe, Tyr, or Val; X₁₅ is Asn, His, Leu, Pro, or Tyr; and X₁₆ is Asn, Asp, His, Phe, Ser, or Tyr; or (E) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-Cys-X₁₆-X₁₇-X₁₈ (SEQ ID NO: 5), wherein X₁ is Arg, Asp, Gly, His, Leu, Phe, Pro, Ser, Trp, Tyr, or is absent; X₂ is Ala, Arg, Asn, Asp, Gly, Pro, Ser, or is absent; X₃ is Arg, Asn, Gln, Glu, Gly, Lys, Met, Pro, Trp or Val; X₅ is Arg, Asn, Gln, Glu, His, Leu, Phe, Pro, Trp, Tyr, or Val; X₆ is Arg, Asp, Gln, Gly, Ile, Lys, Phe, Thr, Trp or Tyr; X₇ is Ala, Arg, Asp, Glu, Gly, Leu, Ser, or Tyr; X₈ is Asp, Gln, Glu, Leu, Met, Phe, Pro, Ser, or Tyr; X₉ is Asp, Leu, Pro, Thr, or Val; X₁₀ is Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp or Tyr; X₁₁ is Ala, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, or Thr; X₁₂ is Ala, Asn, Gln, Gly, Leu, Lys, Phe, Pro, Thr, Trp, or Tyr; X₁₃ is Ala, Arg, Gln, His, Lys, Met, Phe, Pro, Thr, Trp, or Tyr; X₁₄ is Arg, Gln, Glu, Gly, His, Leu, Met, Phe, Pro, Ser, Thr, Tyr, or Val; X₁₆ is Arg, Asp, Gly, His, Lys, Met, Phe, Pro, Ser, or Trp; X₁₇ is Arg, Asn, Asp, Gly, His, Phe, Pro, Ser, Trp or Tyr; and X₁₈ is Ala, Arg, Asn, Asp, His, Leu, Phe, or Trp; or (F) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂ (SEQ ID NO: 6), wherein X₁ is Ala, Arg, Gly, His, Leu, Lys, Met, Phe, Trp, Tyr, or Val; X₂ is Ala, Arg, Gln, His, Ile, Leu, Phe, Thr, Trp, or Tyr; X₃ is Ala, Asp, Lys, Phe, Thr, Trp or Tyr; X₄ is Arg, Asp, Gln, Lys, Met, Phe, Pro, Ser, Tyr, or Val; X₅ is Asp, Leu, Lys, Phe, Pro, Ser, or Val; X₆ is His, Ile, Leu, Pro, Ser, or Thr; X₇ is Arg, Gly, His, Leu, Lys, Met, or Thr; X₈ is Ala, Arg, Asn, Ile, Leu, Lys, Met, or Thr; X₉ is Ala, Asn, Arg, Asp, Glu, Gly, His, Leu, Met, Ser, Trp, Tyr, or Val; X₁₀ is Ile, Leu, Phe, Ser, Thr, Trp, Tyr, or Val; X₁₁ is Ala, Arg, Gly, His, Ile, Leu, Lys, Pro, Ser, Thr, Trp, Tyr, or Val; and X₁₂ is Arg, Asp, His, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val; or (G) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃ (SEQ ID NO: 7), wherein X₁ is Asp, Gln, Glu, Gly, His, Lys, Met, or Trp; X₂ is Arg, Gln, His, Ile, Leu, or Pro; X₃ is Asp, Gly, Ile, Lys, Thr, Tyr or Val; X₄ is Asn, Asp, Gln, Glu, Met, Pro, Ser, or Tyr; X₅ is Asn, Asp, His, Ile, Leu, Met, Pro, Thr or Val; X₆ is Asp, Glu, His, Leu, Lys, Pro, or Val; X₇ is Arg, Asn, Gln, His, Ile, Leu, Met, Pro, or Thr; X₈ is Gln, Gly, His, Leu, Met, Ser, or Thr; X₉ is Asn, Gln, Gly, His, Leu, Lys, Ser, or Thr; X₁₀ is Ala, Gly, Ile, Leu, Lys, Met, or Phe; X₁₁ is Ala, Glu, His, Ile, Leu, Met, Ser, Thr, Trp, Tyr, or Val; X₁₂ is Arg, Gln, Glu, Gly, His, Ile, Lys, Tyr, or Val; and X₁₃ is Arg, Asn, Glu, His, Ile, Ser, Thr, Trp, or Val.
 12. The BLyS binding polypeptide according to claim 11, wherein (a) said polypeptide includes an amino acid sequence of the following formula: X₁-X₂-X₃-Cys-X₅-Phe-X₇-Trp-Glu-Cys-X₁₁-X₁₂-X₁₃ (SEQ ID NO: 1), and the following amino acid positions are independently selected as follows: X₃ is Lys; X₅ is Tyr; X₇ is Pro; X₁₁ is Ala, Gln, His, Phe, or Val; X₁₂ is Asn, Gln, Gly, His, Ser, or Val; X₁₃ is Ala, Asn, Gly, Ile, Pro, or Ser; or combinations of such selections; or (b) said polypeptide includes an amino acid sequence of the following formula: X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-Cys-X₁₂-X₁₃-X₁₄ (SEQ ID NO: 2), and the following amino acid positions are independently selected as follows: X₃ is Asp; X₅ is Asp or Leu; X₆ is Glu or Leu; X₇ is His or Leu; X₈ is Thr or Pro; X₉ is Lys; or combinations of such selections; or (c) said polypeptide includes an amino acid sequence of the following formula: X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys-X₁₃-X₁₄-X₁₅ (SEQ ID NO: 3), and the following amino acid positions are independently selected as follows: X₃ is Ala; X₅ is Asp; X₆ is Ile; X₇ is Val or Leu; X₈is Thr; X₉ is Leu; X₁₁ is Ser; X₁₃ is Val; X₁₅ is Glu or Pro; or combinations of such selections; or (d) said polypeptide includes an amino acid sequence of the following formula: X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-Cys-X₁₄-X₁₅-X₁₆ (SEQ ID NO: 4), and the following amino acid positions are independently selected as follows: X₁ is Ser; X₂ is Arg; X₃ is Asn or Asp; X₇ is Asp; X₈ is Glu or Pro; X₉ is Leu; X₁₀ is Thr; X₁₄ is Leu; X₁₅ is His, Leu, or Pro; X₁₆ is Asp or Ser; or combinations of such selections; or (e) said polypeptide includes an amino acid sequence of the following formula: X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-Cys-X₁₆-X₁₇-X₁₈ (SEQ ID NO: 5), and the following amino acid positions are independently selected as follows: X₁ is Arg; X₂ is Asn, Asp, Gly, or Pro; X₃ is Gly or Met; X₅ is Trp, Tyr, or Val; X₆ is Asp; X₇ is Asp; X₈ is Leu; X₉ is Leu or Thr; X₁₀ is Lys or Thr; X₁₁ is Arg or Leu; X₁₂ is Thr or Trp; X₁₃ is Met or Phe; X₁₄ is Val; X₁₆ is Met; X₁₇ is Arg, His, or Tyr; X₁₈ is Asn or His; or combinations of such selections; or (f) said polypeptide includes an amino acid sequence of the following formula: X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂ (SEQ ID NO: 6), and the following amino acid positions are independently selected as follows: X₁ is Gly, Tyr, or Val; X₂ is His or Tyr; X₃ is Asp or Tyr; X₄ is Asp or Gln; X₅ is Leu or Ser; X₆ is Leu or Thr; X₇ is Lys or Thr; X₈ is Leu or Lys; X₉ is Met or Ser; X₁₀ is Thr or Leu; X₁₁ is Pro or Thr; X₁₂ is Arg or Pro; or combinations of such selections; or (g) said polypeptide includes an amino acid sequence of the following formula: X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃ (SEQ ID NO: 7), and the following amino acid positions are independently selected as follows: X₁ is Glu or Lys; X₂ is His or Pro; X₃ is Tyr; X₄ is Asp or Gln; X₅ is Asn or Thr; X₆ is Asp or Pro; X₇ is Ile or Pro; X₈ is Leu or Thr; X₉ is Lys; X₁₀ is Gly or Met; X₁₁ is Ala or Thr; X₁₂ is Arg or His; X₁₃ is His; or combinations of such selections.
 13. The BLyS binding polypeptide according to claim 11, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-162 as depicted in Tables 1-8.
 14. The BLyS binding polypeptide according to claim 11, comprising an amino acid sequence selected from the group consisting of: AGKEPCYFYWECAVSGPGPEGGGK, (SEQ ID NO:163) AGVPFCDLLTKJICFEAGPGPEGGGK, (SEQ ID NO:164) GSSRLCHMDELTHVCVHFAPPGPEGGGK, (SEQ ID NO:165) GDGGNCYTDSLTKLHFCMGDEPGPEGGGK, (SEQ ID NO:166) GYDVLTKLYFVPGGPGPEGGGK, and (SEQ ID NO:167) WTDSLTGLWFPDGGPGPEGGGK,. (SEQ ID NO:168)


15. A recombinant bacteriophage expressing exogenous DNA encoding a BLyS binding polypeptide comprising an amino acid sequence: Asp-Xaa-Leu-Thr (SEQ ID NO: 446), wherein Xaa is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala.
 16. The bacteriophage according to claim 15, wherein Xaa is Pro or Ser.
 17. The bacteriophage according to claim 15, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: Ala-Gly-Lys-Glu-Pro-Cys-Tyr-Phe-Tyr-Trp-Glu-Cys-Ala-Val-Ser-Gly; (SEQ ID NO:450) Ala-Gly-Val-Pro-Phe-Cys-Asp-Leu-Leu-Thr-Lys-His-Cys-Phe-Glu-Ala-Gly; (SEQ ID NO:451) Gly-Ser-Ser-Arg-Leu-Cys-His-Met-Asp-Glu-Leu-Thr-His-Val-Cys-Val-His-Phe-Ala-Pro; (SEQ ID NO:452) Gly-Asp-Gly-Gly-Asn-Cys-Tyr-Thr-Asp-Ser-Leu-Thr-Lys-Leu-His-Phe-Cys-Met-Gly-Asp-Glu; (SEQ ID NO:453) Gly-Tyr-Asp-Val-Leu-Thr-Lys-Leu-Tyr-Phe-Val-Pro-Gly-Gly; (SEQ ID NO:454) Trp-Thr-Asp-Ser-Leu-Thr-Gly-Leu-Trp-Phe-Pro-Asp-Gly-Gly; (SEQ ID NO:455) Ala-Asn-Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu-Pro-Asp; (SEQ ID NO:186) Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu-Pro-Asp; (SEQ ID NO:456) Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu; (SEQ ID NO:457) Ala-Asn-Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu-Pro-Val; (SEQ ID NO:189) Ala-Asn-Trp-Phe-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu-Pro-Asp; (SEQ ID NO:309) Ala-Asn-Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Ser-Leu-Pro-Asp; (SEQ ID NO:458) Ala-Asn-Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Phe-Pro-Asp; (SEQ ID NO:353) Ala-Asn-Trp-Tyr-Asp-Ser-Leu-Thr-Lys-Leu-Trp-Leu-Pro-Asp. (SEQ ID NO:327)


18. The bacteriophage according to claim 15, wherein said polypeptides comprises the amino acid sequence: X₁-X₂Asp-X₄-Leu-Thr-X₇-Leu-X₉-X₁₀ (SEQ ID NO: 448), wherein ! X₁ is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly, or Ser; X₂ is Tyr, Phe, Glu, Cys, Asn; X₄ is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala; X₇ is Lys, Asn, Gln, Gly, or Arg; X₉ is Trp, Ser, Thr, Arg, Cys, Tyr, or Lys; and X₁₀ is Leu, Phe, Val, Ile, or His.
 19. The bacteriophage according to claim 18, wherein said polypeptide comprises the amino acid sequence: Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu (SEQ ID NO: 436).
 20. The bacteriophage according to claim 18, wherein said polypeptide comprises the amino acid sequence: Ala-X₂-X₃-X₄-Asp-X₆-Leu-Thr-X₉-Leu-X₁₁-X₁₂-X₁₃-X₁₄ (SEQ ID NO: 447), wherein X₂ is any amino acid except Arg; X₃ is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly, or Ser; X₄ is Tyr, Phe, Glu, Cys, Asn; X₆ is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala; X₉ is Lys, Asn, Gln, Gly, or Arg; X₁₁ is Trp, Ser, Thr, Arg, Cys, Tyr, or Lys; X₁₂ is Leu, Phe, Val, Ile, or His; X₁₃ is Pro, Leu, His, Ser, Arg, Asn, Gln, Thr, Val, Ala, Cys, Ile, Phe, or Tyr; and X₁₄ is Asp, Glu, Asn, Val, His, Gln, Arg, Gly, Ser, Tyr, Ala, Cys, Lys, Ile, Thr or Leu.
 21. The bacteriophage according to claim 18, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 186-435 as depicted in Table
 14. 22. A recombinant bacteriophage expressing exogenous DNA encoding a BLyS binding polypeptide comprising an amino acid sequence of the formula: (A) X₁-X₂-X₃-Cys-X₅-Phe-X₇-Trp-Glu-Cys-X₁₁-X₁₂-X₁₃ (SEQ ID NO: 1), wherein X₁ is Ala, Asn, Lys, or Ser; X₂ is Ala, Glu, Met, Ser, or Val; X₃ is Ala, Asn, Lys, or Pro; X₅ is Phe, Trp, or Tyr; X₇ is Pro or Tyr; X₁₁ is Ala, Gln, His, Phe, or Val; X₁₂ is Asn, Gln, Gly, His, Ser, or Val; and X₁₃ is Ala, Asn, Gly, Ile, Pro, or Ser; or (B) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-Cys-X₁₂-X₁₃-X₁₄ (SEQ ID NO: 2), wherein X₁ is Ala, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, or is absent; X₂ is Ala, Asn, Asp, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X₃ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val; X₅ is Asp, Ile, Leu, or Tyr; X₆ is Arg, Asp, Glu, His, Ile, Leu, Lys, Phe, Pro, Tyr, or Val; X₇ is His, Leu, Lys, or Phe; X₈ is Leu, Pro, or Thr; X₉ is Arg, Asn, Gly, His, Ile, Lys, Met, or Trp; X₁₀ is Ala, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Ser, Trp, Tyr, or Val; X₁₂ is Asp, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Ser, Trp, Tyr, or Val; X₁₃ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and X₁₄ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Trp, Tyr, Val, or is absent; or (C) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys-X₁₃-X₁₄-X₁₅ (SEQ ID NO: 3), wherein X₁ is Ala, Arg, Asn, Asp, Leu, Lys, Phe, Pro, Ser, or Thr; X₂ is Asn, Asp, Gln, His, Ile, Lys, Pro, Thr, or Trp; X₃ is Ala, Arg, Asn, Gln, Glu, His, Phe, Pro, or Thr; X₅ is Asn, Asp, Pro, Ser, or Thr; X₆ is Arg, Asp, Ile, Leu, Met, Pro, or Val; X₇ is Ala, Ile, Leu, Pro, Thr, or Val; X₈ is Asn, His, Ile, Leu, Lys, Phe, or Thr; X₉ is Asn, Glu, Gly, His, Leu, Lys, Met, Pro, or Thr; X₁₀ is Arg, Asn, Asp, Gln, Glu, Gly, Ile, Lys, Met, Pro, Ser, or Trp; X₁₁ is Arg, Glu, Gly, Lys, Phe, Ser, Trp, or Tyr; X₁₃ is Gln, Glu, Ile, Leu, Phe, Pro, Ser, Tyr, or Val; X₁₄ is Asn, Gly, Ile, Phe, Pro, Thr, Trp, or Tyr; and X₁₅ is Asn, Asp, Glu, Leu, Lys, Met, Pro, or Thr; or (D) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-Cys-X₁₄-X₁₅-X₁₆ (SEQ ID NO: 4), wherein X₁ is Asn, Asp, His, Leu, Phe, Pro, Ser, Tyr, or is absent; X₂ is Arg, Asn, Asp, His, Phe, Ser, or Trp; X₃ is Asn, Asp, Leu, Pro, Ser, or Val; X₅ is Asp, Gln, His, Ile, Leu, Lys, Met, Phe, or Thr; X₆ is His, Ile, Leu, Met, Phe, Pro, Trp, or Tyr; X₇ is Asp, His, Leu, or Ser; X₈ is Ala, Arg, Asp, Glu, Leu, Phe, Pro, or Thr; X₉ is Ala, Arg, Asn, or Leu; X₁₀ is Ile, Leu, Met, Pro, Ser, or Thr; X₁₁ is Ala, Arg, Asn, Gly, His, Lys, Ser, or Tyr; X₁₂ is Ala, Arg, Asn, Gln, Leu, Met, Ser, Trp, Tyr, or Val; X₁₄ is Asp, Gly, Leu, Phe, Tyr, or Val; X₁₅ is Asn, His, Leu, Pro, or Tyr; and X₁₆ is Asn, Asp, His, Phe, Ser, or Tyr; or (E) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-Cys-X₁₆-X₁₇-X₁₈ (SEQ ID NO: 5), wherein X₁ is Arg, Asp, Gly, His, Leu, Phe, Pro, Ser, Trp, Tyr, or is absent; X₂ is Ala, Arg, Asn, Asp, Gly, Pro, Ser, or is absent; X₃ is Arg, Asn, Gln, Glu, Gly, Lys, Met, Pro, Trp or Val; X₅ is Arg, Asn, Gln, Glu, His, Leu, Phe, Pro, Trp, Tyr, or Val; X₆ is Arg, Asp, Gln, Gly, Ile, Lys, Phe, Thr, Trp or Tyr; X₇ is Ala, Arg, Asp, Glu, Gly, Leu, Ser, or Tyr; X₈ is Asp, Gln, Glu, Leu, Met, Phe, Pro, Ser, or Tyr; X₉ is Asp, Leu, Pro, Thr, or Val; X₁₀ is Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp or Tyr; X₁₁ is Ala, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, or Thr; X₁₂ is Ala, Asn, Gln, Gly, Leu, Lys, Phe, Pro, Thr, Trp, or Tyr; X₁₃ is Ala, Arg, Gln, His, Lys, Met, Phe, Pro, Thr, Trp, or Tyr; X₁₄ is Arg, Gln, Glu, Gly, His, Leu, Met, Phe, Pro, Ser, Thr, Tyr, or Val; X₁₆ is Arg, Asp, Gly, His, Lys, Met, Phe, Pro, Ser, or Trp; X₁₇ is Arg, Asn, Asp, Gly, His, Phe, Pro, Ser, Trp or Tyr; and X₁₈ is Ala, Arg, Asn, Asp, His, Leu, Phe, or Trp; or (F) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂ (SEQ ID NO: 6), wherein X₁ is Ala, Arg, Gly, His, Leu, Lys, Met, Phe, Trp, Tyr, or Val; X₂ is Ala, Arg, Gln, His, Ile, Leu, Phe, Thr, Trp, or Tyr; X₃ is Ala, Asp, Lys, Phe, Thr, Trp or Tyr; X₄ is Arg, Asp, Gln, Lys, Met, Phe, Pro, Ser, Tyr, or Val; X₅ is Asp, Leu, Lys, Phe, Pro, Ser, or Val; X₆ is His, Ile, Leu, Pro, Ser, or Thr; X₇ is Arg, Gly, His, Leu, Lys, Met, or Thr; X₈ is Ala, Arg, Asn, Ile, Leu, Lys, Met, or Thr; X₉ is Ala, Asn, Arg, Asp, Glu, Gly, His, Leu, Met, Ser, Trp, Tyr, or Val; X₁₀ is Ile, Leu, Phe, Ser, Thr, Trp, Tyr, or Val; X₁₁ is Ala, Arg, Gly, His, Ile, Leu, Lys, Pro, Ser, Thr, Trp, Tyr, or Val; and X₁₂ is Arg, Asp, His, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val; or (G) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃ (SEQ ID NO: 7), wherein X₁ is Asp, Gln, Glu, Gly, His, Lys, Met, or Trp; X₂ is Arg, Gln, His, Ile, Leu, or Pro; X₃ is Asp, Gly, Ile, Lys, Thr, Tyr or Val; X₄ is Asn, Asp, Gln, Glu, Met, Pro, Ser, or Tyr; X₅ is Asn, Asp, His, Ile, Leu, Met, Pro, Thr or Val; X₆ is Asp, Glu, His, Leu, Lys, Pro, or Val; X₇ is Arg, Asn, Gln, His, Ile, Leu, Met, Pro, or Thr; X₈ is Gln, Gly, His, Leu, Met, Ser, or Thr; X₉ is Asn, Gln, Gly, His, Leu, Lys, Ser, or Thr; X₁₀ is Ala, Gly, Ile, Leu, Lys, Met, or Phe; X₁₁ is Ala, Glu, His, Ile, Leu, Met, Ser, Thr, Trp, Tyr, or Val; X₁₂ is Arg, Gln, Glu, Gly, His, Ile, Lys, Tyr, or Val; and X₁₃ is Arg, Asn, Glu, His, Ile, Ser, Thr, Trp, or Val.
 23. The recombinant bacteriophage according to claim 22, wherein (a) said polypeptide comprises an amino acid sequence of the following formula: X₁-X₂-X₃-Cys-X₅-Phe-X₇-Trp-Glu-Cys-X₁₁-X₁₂-X₁₃ (SEQ ID NO: 1), and the following amino acid positions are independently selected as follows: X₃ is Lys; X₅ is Tyr; X₇ is Pro; X₁₁ is Ala, Gln, His, Phe, or Val; X₁₂ is Asn, Gln, Gly, His, Ser, or Val; X₁₃ is Ala, Asn, Gly, Ile, Pro, or Ser; or combinations of such selections; or (b) said polypeptide comprises an amino acid sequence of the following formula: X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-Cys-X₁₂-X₁₃-X₁₄ (SEQ ID NO: 2), and the following amino acid positions are independently selected as follows: X₃ is Asp; X₅ is Asp or Leu; X₆ is Glu or Leu; X₇ is His or Leu; X₈ is Thr or Pro; X₉ is Lys; or combinations of such selections; or (c) said polypeptide comprises an amino acid sequence of the following formula: X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys-X₁₃-X₁₄-X₁₅ (SEQ ID NO: 3), and the following amino acid positions are independently selected as follows: X₃ is Ala; X₅ is Asp; X₆ is Ile; X₇ is Val or Leu; X₈ is Thr; X₉ is Leu; X₁₁ is Ser; X₁₃ is Val; X₁₅ is Glu or Pro; or combinations of such selections; or (d) said polypeptide comprises an amino acid sequence of the following formula: X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-Cys-X₁₄-X₁₅-X₁₆ (SEQ ID NO: 4), and the following amino acid positions are independently selected as follows: X₁ is Ser; X₂ is Arg; X₃ is Asn or Asp; X₇ is Asp; X₈ is Glu or Pro; X₉ is Leu; X₁₀ is Thr; X₁₄ is Leu; X₁₅ is His, Leu, or Pro; X₁₆ is Asp or Ser; or combinations of such selections; or (e) said polypeptide comprises an amino acid sequence of the following formula: X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-Cys-X₁₆-X₁₇-X₁₈ (SEQ ID NO: 5), and the following amino acid positions are independently selected as follows: X₁ is Arg; X₂ is Asn, Asp, Gly, or Pro; X₃ is Gly or Met; X₅ is Trp, Tyr, or Val; X₆ is Asp; X₇ is Asp; X₈ is Leu; X₉ is Leu or Thr; X₁₀ is Lys or Thr; X₁₁ is Arg or Leu; X₁₂ is Thr or Trp; X₁₃ is Met or Phe; X₁₄ is Val; X₁₆ is Met; X₁₇ is Arg, His, or Tyr; X₁₈ is Asn or His; or combinations of such selections; or (f) said polypeptide comprises an amino acid sequence of the following formula: X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂ (SEQ ID NO: 6), and the following amino acid positions are independently selected as follows: X₁ is Gly, Tyr, or Val; X₂ is His or Tyr; X₃ is Asp or Tyr; X₄ is Asp or Gln; X₅ is Leu or Ser; X₆ is Leu or Thr; X₇ is Lys or Thr; X₈ is Leu or Lys; X₉ is Met or Ser; X₁₀ is Thr or Leu; X₁₁ is Pro or Thr; X₁₂ is Arg or Pro; or combinations of such selections; or (g) said polypeptide comprises an amino acid sequence of the following formula: X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃ (SEQ ID NO: 7), and the following amino acid positions are independently selected as follows: X₁ is Glu or Lys; X₂ is His or Pro; X₃ is Tyr; X₄ is Asp or Gln; X₅ is Asn or Thr; X₆ is Asp or Pro; X₇ is Ile or Pro; X₈ is Leu or Thr; X₉ is Lys; X₁₀ is Gly or Met; X₁₁ is Ala or Thr; X₁₂ is Arg or His; X₁₃ is His; or combinations of such selections.
 24. A method for detecting BLyS or a BLyS-like polypeptide in a solution suspected of containing it comprising: (a) contacting said solution with a polypeptide according to any of claims 1, 9 or 11, and (b) determining whether binding has occurred between said polypeptide and BLyS or a BLyS-like polypeptide.
 25. A method for purifying BLyS or a BLyS-like polypeptide comprising: (a) immobilizing a binding polypeptide according to any of claims 1, 9 or 11 on a solid support; (b) contacting a solution containing BLyS or a BLyS-like polypeptide with said support; and, thereafter, (c) separating the solution from said support.
 26. BLyS separation media comprising: (a) a chromatographic matrix material, and, immobilized thereon, (b) a BLyS binding molecule comprising a BLyS binding polypeptide as defined in any of claims 1, 9 or
 11. 27. The BLyS separation media according to claim 26, comprising: (a) a chromatographic matrix material, and, immobilized thereon, (b) a BLyS binding molecule comprising a BLyS binding polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-162 and 186-435, as depicted in Tables 1-8 and
 14. 28. A method for separating BLyS or a BLyS-like polypeptide from a solution containing it comprising: (a) contacting said solution with separation media as defined in claim 26, (b) removing unbound material, and (c) eluting bound BLyS or BLyS-like polypeptide from said separation media.
 29. A polynucleotide encoding a BLyS binding polypeptide comprising the amino acid sequence: Asp-Xaa-Leu-Thr (SEQ ID NO: 446), wherein Xaa is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala.
 30. The polynucleotide according to claim 29, wherein Xaa is Pro or Ser.
 31. The polynucleotide according to claim 29, wherein said polypeptides comprises the amino acid sequence: X₁-X₂-Asp-X₄-Leu-Thr-X₇-Leu-X₉-X₁₀ (SEQ ID NO: 448), wherein X₁ is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly, or Ser; X₂ is Tyr, Phe, Glu, Cys, Asn; X₄ is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala; X₇ is Lys, Asn, Gln, Gly, or Arg; X₉ is Trp, Ser, Thr, Arg, Cys, Tyr, or Lys; and X₁₀ is Leu, Phe, Val, Ile, or His.
 32. The polynucleotide according to claim 31, wherein said polypeptide comprises the amino acid sequence: Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu (SEQ ID NO: 436).
 33. The polynucleotide according to claim 31, wherein said polypeptide comprises the amino acid sequence: Ala-X₂-X₃-X₄-Asp-X₆-Leu-Thr-X₉-Leu-X₁₁-X₁₂-X₁₃-X₁₄ (SEQ ID NO: 447), wherein X₂ is any amino acid except Arg; X₃ is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly, or Ser; X₄ is Tyr, Phe, Glu, Cys, Asn; X₆ is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala; X₉ is Lys, Asn, Gln, Gly, or Arg; X₁₁ is Trp, Ser, Thr, Arg, Cys, Tyr, or Lys; X₁₂ is Leu, Phe, Val, Ile, or His; X₁₃ is Pro, Leu, His, Ser, Arg, Asn, Gln, Thr, Val, Ala, Cys, Ile, Phe, or Tyr; and X₁₄ is Asp, Glu, Asn, Val, His, Gln, Arg, Gly, Ser, Tyr, Ala, Cys, Lys, Ile, Thr or Leu.
 34. The polynucleotide according to claim 31, encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 186-435 as depicted in Table
 14. 35. A polynucleotide encoding a BLyS binding polypeptide of the formula: (A) X₁-X₂-X₃-Cys-X₅-Phe-X₇-Trp-Glu-Cys-X₁₁-X₁₂-X₁₃ (SEQ ID NO: 1), wherein X₁ is Ala, Asn, Lys, or Ser; X₂ is Ala, Glu, Met, Ser, or Val; X₃ is Ala, Asn, Lys, or Pro; X₅ is Phe, Trp, or Tyr; X₇ is Pro or Tyr; X₁₁ is Ala, Gln, His, Phe, or Val; X₁₂ is Asn, Gln, Gly, His, Ser, or Val; and X₁₃ is Ala, Asn, Gly, Ile, Pro, or Ser; or (B) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-Cys-X₁₂-X₁₃-X₁₄ (SEQ ID NO: 2), wherein X₁ is Ala, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, or is absent; X₂ is Ala, Asn, Asp, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X₃ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val; X₅ is Asp, Ile, Leu, or Tyr; X₆ is Arg, Asp, Glu, His, Ile, Leu, Lys, Phe, Pro, Tyr, or Val; X₇ is His, Leu, Lys, or Phe; X₈ is Leu, Pro, or Thr; X₉ is Arg, Asn, Gly, His, Ile, Lys, Met, or Trp; X₁₀ is Ala, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Ser, Trp, Tyr, or Val; X₁₂ is Asp, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Ser, Trp, Tyr, or Val; X₁₃ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and X₁₄ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Trp, Tyr, Val, or is absent; or (C) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys-X₁₃-X₁₄-X₁₅ (SEQ ID NO: 3), wherein X₁ is Ala, Arg, Asn, Asp, Leu, Lys, Phe, Pro, Ser, or Thr; X₂ is Asn, Asp, Gln, His, Ile, Lys, Pro, Thr, or Trp; X₃ is Ala, Arg, Asn, Gln, Glu, His, Phe, Pro, or Thr; X₅ is Asn, Asp, Pro, Ser, or Thr; X₆ is Arg, Asp, Ile, Leu, Met, Pro, or Val; X₇ is Ala, Ile, Leu, Pro, Thr, or Val; X₈ is Asn, His, Ile, Leu, Lys, Phe, or Thr; X₉ is Asn, Glu, Gly, His, Leu, Lys, Met, Pro, or Thr; X₁₀ is Arg, Asn, Asp, Gln, Glu, Gly, Ile, Lys, Met, Pro, Ser, or Trp; X₁₁ is Arg, Glu, Gly, Lys, Phe, Ser, Trp, or Tyr; X₁₃ is Gln, Glu, Ile, Leu, Phe, Pro, Ser, Tyr, or Val; X₁₄ is Asn, Gly, Ile, Phe, Pro, Thr, Trp, or Tyr; and X₁₅ is Asn, Asp, Glu, Leu, Lys, Met, Pro, or Thr; or (D) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉X₁₀-X₁₁-X₁₂-Cys-X₁₄-X₁₅-X₁₆ (SEQ ID NO: 4), wherein X₁ is Asn, Asp, His, Leu, Phe, Pro, Ser, Tyr, or is absent; X₂ is Arg, Asn, Asp, His, Phe, Ser, or Trp; X₃ is Asn, Asp, Leu, Pro, Ser, or Val; X₅ is Asp, Gln, His, Ile, Leu, Lys, Met, Phe, or Thr; X₆ is His, Ile, Leu, Met, Phe, Pro, Trp, or Tyr; X₇ is Asp, His, Leu, or Ser; X₈ is Ala, Arg, Asp, Glu, Leu, Phe, Pro, or Thr; X₉ is Ala, Arg, Asn, or Leu; X₁₀ is Ile, Leu, Met, Pro, Ser, or Thr; X₁₁ is Ala, Arg, Asn, Gly, His, Lys, Ser, or Tyr; X₁₂ is Ala, Arg, Asn, Gln, Leu, Met, Ser, Trp, Tyr, or Val; X₁₄ is Asp, Gly, Leu, Phe, Tyr, or Val; X₁₅ is Asn, His, Leu, Pro, or Tyr; and X₁₆ is Asn, Asp, His, Phe, Ser, or Tyr; or (E) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-Cys-X₁₆-X₁₇-X₁₈ (SEQ ID NO: 5), wherein X₁ is Arg, Asp, Gly, His, Leu, Phe, Pro, Ser, Trp, Tyr, or is absent; X₂ is Ala, Arg, Asn, Asp, Gly, Pro, Ser, or is absent; X₃ is Arg, Asn, Gln, Glu, Gly, Lys, Met, Pro, Trp or Val; X₅ is Arg, Asn, Gln, Glu, His, Leu, Phe, Pro, Trp, Tyr, or Val; X₆ is Arg, Asp, Gln, Gly, Ile, Lys, Phe, Thr, Trp or Tyr; X₇ is Ala, Arg, Asp, Glu, Gly, Leu, Ser, or Tyr; X₈ is Asp, Gln, Glu, Leu, Met, Phe, Pro, Ser, or Tyr; X₉ is Asp, Leu, Pro, Thr, or Val; X₁₀ is Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp or Tyr; X₁₁ is Ala, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, or Thr; X₁₂ is Ala, Asn, Gln, Gly, Leu, Lys, Phe, Pro, Thr, Trp, or Tyr; X₁₃ is Ala, Arg, Gln, His, Lys, Met, Phe, Pro, Thr, Trp, or Tyr; X₁₄ is Arg, Gln, Glu, Gly, His, Leu, Met, Phe, Pro, Ser, Thr, Tyr, or Val; X₁₆ is Arg, Asp, Gly, His, Lys, Met, Phe, Pro, Ser, or Trp; X₁₇ is Arg, Asn, Asp, Gly, His, Phe, Pro, Ser, Trp or Tyr; and X₁₈ is Ala, Arg, Asn, Asp, His, Leu, Phe, or Trp; or (F) X₁-X₂₋X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂ (SEQ ID NO: 6), wherein X₁ is Ala, Arg, Gly, His, Leu, Lys, Met, Phe, Trp, Tyr, or Val; X₂ is Ala, Arg, Gln, His, Ile, Leu, Phe, Thr, Trp, or Tyr; X₃ is Ala, Asp, Lys, Phe, Thr, Trp or Tyr; X₄ is Arg, Asp, Gln, Lys, Met, Phe, Pro, Ser, Tyr, or Val; X₅ is Asp, Leu, Lys, Phe, Pro, Ser, or Val; X₆ is His, Ile, Leu, Pro, Ser, or Thr; X₇ is Arg, Gly, His, Leu, Lys, Met, or Thr; X₈ is Ala, Arg, Asn, Ile, Leu, Lys, Met, or Thr; X₉ is Ala, Asn, Arg, Asp, Glu, Gly, His, Leu, Met, Ser, Trp, Tyr, or Val; X₁₀ is Ile, Leu, Phe, Ser, Thr, Trp, Tyr, or Val; X₁₁ is Ala, Arg, Gly, His, Ile, Leu, Lys, Pro, Ser, Thr, Trp, Tyr, or Val; and X₁₂ is Arg, Asp, His, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val; or (G) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃ (SEQ ID NO: 7), wherein X₁ is Asp, Gln, Glu, Gly, His, Lys, Met, or Trp; X₂ is Arg, Gln, His, Ile, Leu, or Pro; X₃ is Asp, Gly, Ile, Lys, Thr, Tyr or Val; X₄ is Asn, Asp, Gln, Glu, Met, Pro, Ser, or Tyr; X₅ is Asn, Asp, His, Ile, Leu, Met, Pro, Thr or Val; X₆ is Asp, Glu, His, Leu, Lys, Pro, or Val; X₇ is Arg, Asn, Gln, His, Ile, Leu, Met, Pro, or Thr; X₈ is Gln, Gly, His, Leu, Met, Ser, or Thr; X₉ is Asn, Gln, Gly, His, Leu, Lys, Ser, or Thr; X₁₀ is Ala, Gly, Ile, Leu, Lys, Met, or Phe; X₁₁ is Ala, Glu, His, Ile, Leu, Met, Ser, Thr, Trp, Tyr, or Val; X₁₂ is Arg, Gln, Glu, Gly, His, Ile, Lys, Tyr, or Val; and X₁₃ is Arg, Asn, Glu, His, Ile, Ser, Thr, Trp, or Val.
 36. A method for identifying a binding molecule for a BLyS target comprising the steps of utilizing a BLyS binding polypeptide according to any of claims 1, 9 or 11 to form a complex with BLyS or a BLyS-like polypeptide, contacting said complex with one or more potential BLyS target binding molecules, and determining whether said one or more potential BLyS target binding molecules competes with said BLyS binding polypeptide to form a complex with said BLyS or BLyS-like polypeptide.
 37. A BLyS affinity maturation library, comprising a population of at least 10³ polypeptides, wherein the polypeptides of said population comprise the amino acid sequence: Ala-X₂-X₃-X₄-Asp-X₆-Leu-Thr-X₉-Leu-X₁₁-X₁₂-X₁₃-X₁₄ (SEQ ID NO: 449), wherein X₂ is any amino acid; X₃ is any amino acid; X₄ is any amino acid; X₆ is any amino acid; X₉ is any amino acid; X₁₁ is any amino acid; X₁₂ is any amino acid; X₁₃ is any amino acid; and X₁₄ is any amino acid.
 38. A DNA template encoding a multiplicity of BLyS binding polypeptides, comprising the sequence: GCT NNN NNN NNN GAT NNN CTT ACT NNN CTC NNN NNN NNN NNN (SEQ ID NO: 185). 